Detection of genome editing

ABSTRACT

Methods, compositions, and kits are provided for quantification of genome editing.

CROSS-REFERENCE TO RELATED PATENT APPLICATIONS

This application is a continuation of U.S. application Ser. No.16/352,600, filed Mar. 13, 2019, which is a continuation of U.S.application Ser. No. 14/991,818 filed Jan. 8, 2016, now U.S. Pat. No.10,280,451, which claims priority to U.S. Provisional Application No.62/101,828, filed Jan. 9, 2015 and U.S. Provisional Application No.62/201,446, filed Aug. 5, 2015, each of which is incorporated herein byreference in its entirety.

STATEMENT AS TO RIGHTS TO INVENTIONS MADE UNDER FEDERALLY SPONSOREDRESEARCH AND DEVELOPMENT

This invention was made with government support under grants HL100406and HL098179 awarded by the National Institutes of Health. Thegovernment has certain rights in this invention.

REFERENCE TO SUBMISSION OF A SEQUENCE LISTING

This application includes a Sequence Listing as an ASCII text file named“094868-1290571-108640US_SL.txt” created Dec. 17, 2021, and containing11,138 bytes. The material contained in this text file is incorporatedby reference in its entirety for all purposes.

BACKGROUND OF THE INVENTION

Methods and compositions for genome editing are of great interest to thebiotechnology and pharmaceutical community. Indeed, the ability toprecisely edit genomes can be considered a rate limiting step for thedevelopment of a wide variety of therapeutic applications. Recenttechnologies have expanded the number of tools available for genomeediting. Such tools include zinc finger nucleases (See, e.g., Kim etal., Proc Natl Acad Sci USA. 1996 Feb. 6; 93(3):1156-60); transcriptionactivator-like effector nucleases (TALENS) (See, e.g., Miller et al.,Nat Biotechnol. 2011 February; 29(2):143-8); CRISPR-Cas (See, e.g., Maliet al., Nat Methods. 2013 October; 10(10):957-63) nucleases; and nickaseversions thereof.

BRIEF SUMMARY OF THE INVENTION

In one aspect, the present invention provides a method for simultaneousquantification of homology directed repair (HDR) and non-homologous endjoining (NHEJ) genome editing products in a sample of cells, whereinsaid sample of cells has been contacted with a site-specific genomeediting reagent configured to cleave or nick DNA in a target genomicregion and an HDR template nucleic acid, or the sample of cellscomprises cells that have been contacted with a site-specific genomeediting reagent configured to cleave or nick DNA in a target genomicregion the method comprising: a) forming a plurality of mixturepartitions having an average volume, wherein the mixture partitionscomprise: i) the target genomic region; ii) a DNA-dependent DNApolymerase; iii) a forward and a reverse oligonucleotide amplificationprimer, wherein the forward and reverse primers hybridize to oppositestrands of, and flank, the target genomic region, and wherein theprimers are configured to amplify the target genomic region in thepresence of the polymerase; iv) a detectably labeled oligonucleotidereference probe, wherein the reference probe hybridizes to a wild-typetarget genomic region, a target genomic region containing an HDRmutation introduced by homology directed repair of DNA damage from thesite specific genome editing reagent, and a target genomic regioncontaining an NHEJ mutation introduced by NHEJ repair of DNA damage fromthe site specific genome editing reagent; v) a detectably labeledoligonucleotide HDR probe, wherein the HDR probe hybridizes to thetarget genomic region containing the HDR mutation; and vi) a detectablylabeled oligonucleotide NHEJ drop-off probe, wherein the NHEJ drop-offprobe hybridizes to the wild-type target genomic region, and wherein theNHEJ drop-off probe does not hybridize to the target genomic regioncontaining the NHEJ mutation; b) amplifying the target genomic region inthe plurality of mixture partitions; and c) determining a quantity ofwild-type target genomic regions, a quantity of target genomic regionscontaining an HDR mutation, and a quantity of target genomic regionscontaining an NHEJ mutation by detecting hybridization of the labeledprobes to the target genomic regions in the plurality of mixturepartitions.

In some embodiments, the sample of cells comprises cells that have beencontacted with a site-specific genome editing reagent configured tocleave or nick DNA in a target genomic region and an HDR templatenucleic acid. In some cases, at least a portion of the contacted cellshave been edited by insertion of the HDR template nucleic acid into acleavage site of the site-specific genome editing reagent via homologydirected repair. In some embodiments, the sample of cells comprisescells that have been contacted with a site-specific genome editingreagent configured to cleave or nick DNA in a target genomic region(e.g., without contacting with an HDR template nucleic acid). In somecases, at least a portion of the contacted cells have been edited bynon-homologous end joining repair at a cleavage site of thesite-specific genome editing reagent.

In some embodiments, the reference and HDR probes comprise the samedetectable label. In some embodiments, the HDR probe does not hybridizeto the wild-type target genomic region. In some embodiments, the NHEJdrop off probe does not hybridize to the target genomic regioncontaining the HDR mutation. In some embodiments, the NHEJ drop offprobe hybridizes to the target genomic region containing the HDRmutation. In some embodiments, the DNA dependent DNA polymerasecomprises 5′ to 3′ exonuclease activity and c) comprises detecting anincrease in fluorescence caused by 5′ to 3′ exonuclease digestion of thehybridized labeled probes in the plurality of mixture partitions. Insome embodiments, the plurality of mixture partitions further comprisean HDR dark oligonucleotide probe, wherein the HDR dark probe comprisesa 3′ end that is not extendable by the DNA polymerase, and wherein theHDR dark probe competes for hybridization of the HDR probe to thewild-type target genomic region.

In some embodiments, the NHEJ drop-off probe competes for hybridizationof the HDR probe to the wild-type target genomic region. In someembodiments, the site-specific genome editing reagent is a CRISPR-Cas9reagent, a TALEN, or a Zinc-Finger Nuclease. In some embodiments, thesite-specific genome editing reagent comprises two site-specific genomenickases, wherein each nickase is configured to nick target DNA in thegenome of a cell within less than about 1 kb of the other nickase. Insome embodiments, the method comprises, before the forming the pluralityof mixture partitions, providing the sample of cells that has beencontacted with a site-specific genome editing reagent and extracting thetarget genomic regions. In some embodiments, the method comprises,before the forming the plurality of mixture partitions, providing thesample of cells and extracting the target genomic regions.

In some cases, the providing comprises contacting a plurality of cellswith the site-specific genome editing reagent. In some cases, theplurality of cells contacted with the site-specific genome editingreagent are administered to a patient, and the providing comprisesproviding a sample of cells from the patient to which the cells havebeen administered. In some cases, the providing further comprisescontacting the plurality of cells with an HDR template nucleic acid,wherein the HDR template nucleic acid comprises a mutation of a portionof the target genomic region and is configured to function as a templateduring homology directed repair of the target genomic region and therebyintroduce the HDR mutation into the target genomic region. In somecases, the HDR template nucleic acid comprises DNA. In some cases, theHDR template nucleic acid is a single stranded DNA oligonucleotide.

In some embodiments, the plurality of mixture partitions comprise aplurality of structurally different detectably labeled oligonucleotideNHEJ drop-off probes, wherein the plurality of structurally differentNHEJ drop-off probes hybridize to different sub-regions of the wild-typetarget genomic region. In some cases, the plurality of structurallydifferent NHEJ drop off probes do not hybridize to NHEJ mutatedsub-regions. In some cases, wherein the plurality of structurallydifferent NHEJ drop off probes do not hybridize to NHEJ mutatedsub-regions and do not hybridize to HDR mutated sub-regions. In somecases, wherein the plurality of structurally different NHEJ drop offprobes do not hybridize to NHEJ mutated sub-regions and do hybridize totarget genomic regions containing an HDR mutation.

In some embodiments, c) comprises determining a concentration of targetgenomic regions having the NHEJ mutation in the sample by detecting andcounting: i) a total number of mixture partitions that do not containthe target genomic region; and ii) a total number of—mixture partitionsin which only the target genomic region containing the NHEJ mutation isdetected; and—mixture partitions that do not contain the target genomicregion, wherein the concentration is calculated as a reciprocal of theaverage volume of the plurality of mixture partitions multiplied by anegative natural log of a ratio of i) divided by ii).

In some embodiments, c) comprises determining a concentration of targetgenomic regions having the HDR mutation in the sample by detecting andcounting: i) a total number of:—mixture partitions that do not containthe target genomic region;—mixture partitions in which only the targetgenomic region containing the NHEJ mutation is detected;—mixturepartitions in which only the wild-type target genomic region isdetected; and—mixture partitions in which only the wild-type targetgenomic region and the target genomic region containing the NHEJmutation are detected; and ii) a total number of—i); and—mixturepartitions in which the HDR mutation is detected, wherein theconcentration is calculated as the reciprocal of an average volume ofthe plurality of mixture partitions multiplied by a negative natural logof a ratio of i) divided by ii).

In some embodiments, c) comprises determining a concentration ofwild-type target genomic regions in the sample by detecting andcounting: i) a total number of:—mixture partitions that do not containthe target genomic region; and—mixture partitions in which only thetarget genomic region containing the NHEJ mutation is detected; and ii)a total number of—i);—mixture partitions in which only the wild-typetarget genomic region is detected; and—mixture partitions in which onlythe wild-type target genomic region and the target genomic regioncontaining the NHEJ mutation are detected; and wherein the concentrationis calculated as the reciprocal of an average volume of the plurality ofmixture partitions multiplied by a negative natural log of a ratio of i)divided by ii).

In another aspect, the present invention provides a method foridentifying an optimized condition for genome editing of a cell, themethod comprising: a) performing site specific genome editing of aplurality of cells under a first set of conditions to provide firstsample of cells; b) performing site specific genome editing of aplurality of cells under a second set of conditions to provide a secondsample of cells; c) performing any of the methods described above orelsewhere herein to quantify a number of NHEJ edited target genomicregions and HDR edited target genomic regions in the first and secondsamples of cells to determine a genome editing efficiency for the firstand second set of conditions; d) comparing the genome editing efficiencyof the first and second set of conditions to identify a set ofconditions that provides a higher genome editing efficiency; and e)selecting the set of conditions that provides higher genome editingefficiency as the optimized condition for genome editing.

In some embodiments, a) comprises contacting the first sample of cellswith a first concentration of genome editing reagent; and b) comprisescontacting the second sample of cells with a second concentration ofgenome editing reagent. In some embodiments, a) comprises contacting thefirst sample of cells with a first genome editing reagent; and b)comprises contacting the second sample of cells with a secondstructurally different genome editing reagent. In some embodiments, c)comprises determining an HDR genome editing efficiency for the first setof conditions and determining an HDR genome editing efficiency for thesecond set of conditions; d) comprises comparing the HDR genome editingefficiency of the first and second set of conditions to identify a setof conditions that provides a higher HDR genome editing efficiency; ande) comprises selecting the set of conditions that provides higher HDRgenome editing efficiency as the optimized condition for genome editing.

In some cases, c) comprises determining an NHEJ genome editingefficiency for the first set of conditions and determining an NHEJgenome editing efficiency for the second set of conditions; d) comprisescomparing the NHEJ genome editing efficiency of the first and second setof conditions to identify a set of conditions that provides a lower NHEJgenome editing efficiency; and e) comprises selecting the set ofconditions that provides higher HDR genome editing efficiency and alower NHEJ genome editing efficiency as the optimized condition forgenome editing.

In some embodiments, of one of the foregoing methods, the sample ofcells is from a patient to whom genome edited cells have beenadministered and the method further comprises estimating a number ofgenome edited cells in the patient from a determined quantity ofwild-type target genomic regions, a determined quantity of targetgenomic regions containing an HDR mutation, and/or a determined quantityof target genomic regions containing an NHEJ mutation.

In one aspect, the present invention provides a method of monitoring apopulation of cells in a patient, wherein the cells comprise apopulation of edited genomes (e.g., a clonal population of editedgenomes), the method comprising: providing a sample from an individualto whom the cells comprising the population of edited genomes have beenadministered (e.g., as a component of a treatment); analyzing the sampleto determine a number of edited genomes or fragments thereof in thesample; and estimating the number of cells comprising the population ofedited genomes (e.g., clonal population of edited genomes) in thepatient from the number of edited genomes or fragments thereof in thesample. In some embodiments, the analyzing the sample to determine thenumber of edited genomes or fragments thereof comprises quantifyinghomology directed repair (HDR), non-homologous end joining (NHEJ),simultaneous HDR and NHEJ, and/or the number of wild-type (e.g.,unedited) genomes or fragments thereof in the sample.

In some cases, the method comprises analyzing the sample to determinethe number of edited genomes or fragments thereof by quantifying HDR,NHEJ, or HDR and NHEJ using any one of the foregoing HDR and/or NHEJquantification methods. In some cases, the analyzing the sample todetermine the number of edited genomes or fragments thereof comprisesquantifying a number of genomes containing a polymorphism that isinduced by genome editing. In some cases, the quantifying comprisesdroplet digital nucleic acid amplification. In some cases, the analyzingthe sample to determine the number of edited genomes or fragmentsthereof comprises massively parallel sequencing, high throughputsequencing, or single molecule sequencing.

In some cases, one of the foregoing methods further comprisesdetermining whether to administer additional cells comprising thepopulation of edited genomes (e.g., the clonal population of editedgenomes) to the patient based on the estimated the number of cellscomprising the population of edited genomes in the patient. In somecases, one of the foregoing methods further comprises comparing theestimated number of cells comprising the (e.g., clonal) population ofedited genomes in the patient to a number of genome edited cells thathave been administered to the patient. In some cases, a differencebetween the number of cells comprising the (e.g., clonal) population ofedited genomes in the patient and the number of cells that have beenadministered to the patient indicates whether the treatment is or willbe successful. In some cases, the method comprises determining a riskfactor (e.g., odds ratio) for the success or failure of the treatmentbased on a change in the number of cells comprising the (e.g., clonal)population of edited genomes in the patient from administration of thecells to obtaining the sample from the patient.

In some cases, if the number of cells comprising the (e.g., clonal)population of edited genomes in the patient from administration of thecells to obtaining the sample from the patient decreases, orsubstantially decreases (e.g., decreases by at least about 25%, 50%,75%, 90%, or more), then the method comprises determining to administeradditional cells comprising the (e.g., clonal) population of editedgenomes to the patient. In some cases a decrease, or substantialdecrease (e.g., a decrease of at least about 25%, 50%, 75%, 90%, ormore), in the number of cells comprising the (e.g., clonal) populationof edited genomes in the patient from administration of the cells toobtaining the sample from the patient indicates that the treatment willnot be successful. In some cases, an increase or substantially nodecrease in the in the number of cells comprising the (e.g., clonal)population of edited genomes in the patient from administration of thecells to obtaining the sample from the patient indicates that thetreatment will be successful. In some cases, a decrease or substantialdecrease, in the number of cells comprising the (e.g., clonal)population of edited genomes in the patient from administration of thecells to obtaining the sample from the patient indicates theadministration of a drug or chemotherapeutic agent to the patient, orindicates that an adjustment (e.g., a change in dose or a change in thedrug administered) in a drug regimen.

In some cases, the method comprises determining whether to administeradditional cells comprising the (e.g., clonal) population of editedgenomes to the patient based on a change in the number of cellscomprising the (e.g., clonal) population of edited genomes in thepatient from administration of the cells to obtaining the sample fromthe patient. In some cases, a decrease in the number of cells comprisingthe (e.g., clonal) population of edited genomes in the patient fromadministration of the cells to obtaining the sample from the patientindicates the need to administer additional cells comprising the (e.g.,clonal) population of edited genomes to the patient. In some cases, themethod comprises determining whether to administer cells comprising asecond (e.g., structurally distinct) population (e.g., clonalpopulation) of alternatively edited genomes to the patient based on achange in the number of cells comprising the (e.g., clonal) populationof edited genomes in the patient from administration of the cells toobtaining the sample from the patient.

In some embodiments, the method further comprises: analyzing a secondsample to estimate a second number of cells comprising the (e.g.,clonal) population of edited genomes in the patient, wherein the secondsample is a sample that has been taken from the patient at a later pointin time than the first sample; and comparing the change in the number ofcells comprising the (e.g., clonal) population of edited genomes in thepatient from the first to the second sample. In some cases, the changein the number of genome edited cells indicates whether the treatment isor will be successful. In some cases, a decrease (e.g., a substantialdecrease of at least 25%, 50%, 75%, 90%, or more) in the number of cellscomprising the (e.g., clonal) population of edited genomes in thepatient from the first to the second sample indicates that the treatmentis or will be unsuccessful. In some cases, an increase (e.g., asubstantial increase of at least2 5%, 50%, 75%, 90%, or more) in thenumber of cells comprising the (e.g., clonal) population of editedgenomes in the patient from the first to the second sample indicatesthat the treatment is or will be successful. In some cases a rate ofincrease or decrease in the number of cells comprising the (e.g.,clonal) population of edited genomes in the patient from the first tothe second sample indicates that the treatment is or will be successfulor unsuccessful respectively.

In some cases, the method comprises determining a risk factor for thesuccess or failure of the treatment based on the change (e.g., increaseor decrease) or rate of change in the number of cells comprising the(e.g., clonal) population of edited genomes in the patient from thefirst to the second sample. In some cases, the risk factor is an oddsratio.

In some embodiments, the method comprises determining whether toadminister additional cells comprising the (e.g., clonal) population ofedited genomes to the patient based on the change in the number of cellscomprising the (e.g., clonal) population of edited genomes in thepatient from the first to the second sample. In some cases, a decreasein the number of cells comprising the (e.g., clonal) population ofedited genomes in the patient from the first to the second sampleindicates the need to administer additional cells comprising the (e.g.,clonal) population of edited genomes to the patient. In some cases, themethod comprises determining whether to administer cells comprising asecond (e.g., structurally distinct) population (e.g., clonalpopulation) of alternatively edited genomes to the patient based on achange in the number of cells comprising the clonal population of editedgenomes in the patient from the first to the second sample. For example,a decrease in the number of genome edited cells from the first to thesecond sample can indicate a need to administer a second population ofalternatively edited genomes to the patient.

In another aspect the present invention provides a method of monitoringa population of cells in vitro, wherein the cells comprise a populationof edited genomes (e.g., a clonal population of edited genomes), themethod comprising: providing a population of cells comprising apopulation of edited genomes (e.g., clonal population of editedgenomes), quantifying a first number of edited genomes in a sample ofthe population, selecting the population of cells, quantifying a secondnumber of edited genomes in a sample of the population, and comparingthe first and second numbers to determine a response to the selection.In some embodiments, the quantifying comprises quantifying homologydirected repair (HDR), non-homologous end joining (NHEJ), simultaneousHDR and NHEJ, and/or the number of wild-type (e.g., unedited) genomes orfragments thereof in the sample.

In some cases, the selecting comprises contacting the population ofcells with a drug or chemotherapeutic. In some cases, the selectingcomprises contacting the population of cells with a growth factor orcytokine. In some cases, the selecting comprises culturing the cells orpassaging the cells.

In some cases, the quantifying comprises quantifying HDR, NHEJ, or HDRand NHEJ using any one of the foregoing HDR and/or NHEJ quantificationmethods. In some cases, the the quantifying comprises quantifying anumber of genomes containing a polymorphism that is induced by genomeediting. In some cases, the quantifying comprises droplet digitalnucleic acid amplification. In some cases, the quantifying comprisesmassively parallel sequencing, high throughput sequencing, or singlemolecule sequencing.

In another aspect the present invention provides a method of generatinga specified dose of genome edited cells, the method comprising:providing a population of cells comprising a population of editedgenomes (e.g., clonal population of edited genomes), quantifying anumber of edited genomes in a sample of the population, mixing thepopulation of cells comprising the population of edited genomes with apopulation of (e.g., wild-type) cells that have not been edited by asite specific genome editing reagent, to obtain a population of cellsthat has the specified dose of genome edited cells. In some cases, thespecified dose of genome edited cells is a dose containing, orcontaining about, 1%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, or 75%,genome edited cells. In some cases, the specified dose of genome editedcells is a dose containing, or containing from about, 20% genome editedcells to about 50% genome edited cells.

Definitions

Unless defined otherwise, all technical and scientific terms used hereingenerally have the same meaning as commonly understood by one ofordinary skill in the art to which this invention belongs. Generally,the nomenclature used herein and the laboratory procedures in cellculture, molecular genetics, organic chemistry, and nucleic acidchemistry and hybridization described below are those well-known andcommonly employed in the art. Standard techniques are used for nucleicacid synthesis. The techniques and procedures are generally performedaccording to conventional methods in the art and various generalreferences (see generally, Sambrook et al. MOLECULAR CLONING: ALABORATORY MANUAL, 2d ed. (1989) Cold Spring Harbor Laboratory Press,Cold Spring Harbor, N.Y., which is incorporated herein by reference),which are provided throughout this document.

The term “amplification reaction” refers to any in vitro means formultiplying the copies of a target sequence of nucleic acid in a linearor exponential manner. Such methods include but are not limited topolymerase chain reaction (PCR); DNA ligase chain reaction (see U.S.Pat. Nos. 4,683,195 and 4,683,202; PCR Protocols: A Guide to Methods andApplications (Innis et al., eds, 1990)) (LCR); QBeta RNA replicase andRNA transcription-based amplification reactions (e.g., amplificationthat involves T7, T3, or SP6 primed RNA polymerization), such as thetranscription amplification system (TAS), nucleic acid sequence basedamplification (NASBA), and self-sustained sequence replication (3 SR);isothermal amplification reactions (e.g., single-primer isothermalamplification (SPIA)); as well as others known to those of skill in theart.

“Amplifying” refers to a step of submitting a solution to conditionssufficient to allow for amplification of a target polynucleotide if allof the components of the reaction are intact. Components of anamplification reaction include, e.g., one or more primers, apolynucleotide template, polymerase, nucleotides, and the like. The term“amplifying” typically refers to an exponential increase in targetnucleic acid. However, “amplifying” as used herein can also refer tolinear increases in the numbers of a select target sequence of nucleicacid, such as is obtained with cycle sequencing or linear amplification.Amplifying produces an amplification product, or “amplicon.”

The term “amplification reaction mixture” refers to an aqueous solutioncomprising the various reagents used to amplify a target nucleic acid.These include enzymes, aqueous buffers, salts, amplification primers,target nucleic acid, and nucleoside triphosphates. Amplificationreaction mixtures may also further include stabilizers and otheradditives to optimize efficiency and specificity. Depending upon thecontext, the mixture can be either a complete or incompleteamplification reaction mixture

“Polymerase chain reaction” or “PCR” refers to a method whereby aspecific segment or subsequence of a target double-stranded DNA, isamplified in a geometric progression. PCR is well known to those ofskill in the art; see, e.g., U.S. Pat. Nos. 4,683,195 and 4,683,202; andPCR Protocols: A Guide to Methods and Applications, Innis et al., eds,1990. Exemplary PCR reaction conditions typically comprise either two orthree step cycles. Two step cycles have a denaturation step followed bya hybridization/elongation step. Three step cycles comprise adenaturation step followed by a hybridization step followed by aseparate elongation step.

A “primer” refers to a polynucleotide sequence that hybridizes to asequence on a target nucleic acid and serves as a point of initiation ofnucleic acid synthesis. Primers can be of a variety of lengths and areoften less than 50 nucleotides in length, for example 12-30 nucleotides,in length. The length and sequences of primers for use in PCR can bedesigned based on principles known to those of skill in the art, see,e.g., Innis et al., supra. Primers can be DNA, RNA, or a chimera of DNAand RNA portions. In some cases, primers can include one or moremodified or non-natural nucleotide bases. In some cases, primers arelabeled. Primers that prime the amplification (e.g., PCR) of a targetpolynucleotide sequence are referred to as “amplification primers.”

The term “probe” refers to a molecule (e.g., a protein, nucleic acid,aptamer, etc.) that specifically interacts with or specifically bindsto, and thus detects, a target polynucleotide. Non-limiting examples ofmolecules that specifically interact with or specifically bind to atarget polynucleotide include nucleic acids (e.g., oligonucleotides),proteins (e.g., antibodies, transcription factors, zinc finger proteins,non-antibody protein scaffolds, etc.), and aptamers. Generally, theprobe is labeled with a detectable label. The probe can indicate thepresence or level of the target polynucleotide by either an increase ordecrease in signal from the detectable label. In some cases, the probesdetect the target polynucleotide in an amplification reaction by beingdigested by the 5′ to 3′ exonuclease activity of a DNA dependent DNApolymerase.

Exemplary probes include oligonucleotide primers having hairpinstructures with a fluorescent molecule held in proximity to afluorescent quencher until forced apart by primer extension, e.g.,Whitecombe et al., Nature Biotechnology, 17: 804-807 (1999)(AMPLIFLUOR™, hairpin primers). Exemplary probes may alternativelycomprise an oligonucleotide attached to a fluorophore and a fluorescencequencher, wherein the fluorophore and quencher are in proximity untilthe oligonucleotide specifically binds to an amplification product, e.g.Gelfand et al., U.S. Pat. No. 5,210,015 (TAQMAN™, PCR probes); Nazarenkoet al., Nucleic Acids Research, 25: 2516-2521 (1997) (“scorpionprobes”); and Tyagi et al., Nature Biotechnology, 16: 49-53 (1998)(“molecular beacons”). Such probes may be used to measure the totalamount of reaction product at the completion of a reaction or to measurethe generation of amplification product during an amplificationreaction.

The terms “label,” “detectable label, “detectable moiety,” and liketerms refer to a composition detectable by spectroscopic, photochemical,biochemical, immunochemical, chemical, or other physical means. Forexample, useful labels include fluorescent dyes (fluorophores),luminescent agents, electron-dense reagents, enzymes (e.g., as commonlyused in an ELISA), biotin, digoxigenin, ³²P and other isotopes, haptens,and proteins which can be made detectable, e.g., by incorporating aradiolabel into the peptide or used to detect antibodies specificallyreactive with the peptide. The term includes combinations of singlelabeling agents, e.g., a combination of fluorophores that provides aunique detectable signature, e.g., at a particular wavelength orcombination of wavelengths. Any method known in the art for conjugatinglabel to a desired agent may be employed, e.g., using methods describedin Hermanson, Bioconjugate Techniques 1996, Academic Press, Inc., SanDiego.

A nucleic acid, or a portion thereof, “hybridizes” to another nucleicacid under conditions such that non-specific hybridization is minimal ata defined temperature in a physiological buffer (e.g., pH 6-9, 25-150 mMchloride salt). In some cases, a nucleic acid, or portion thereof,hybridizes to a conserved sequence shared among a group of targetnucleic acids. In some cases, a primer, or portion thereof, canhybridize to a primer binding site if there are at least about 6, 8, 10,12, 14, 16, or 18 contiguous complementary nucleotides, including“universal” nucleotides that are complementary to more than onenucleotide partner. Alternatively, a primer, or portion thereof, canhybridize to a primer binding site if there are fewer than 1 or 2complementarity mismatches over at least about 12, 14, 16, or 18contiguous complementary nucleotides. In some embodiments, the definedtemperature at which specific hybridization occurs is room temperature.In some embodiments, the defined temperature at which specifichybridization occurs is higher than room temperature. In someembodiments, the defined temperature at which specific hybridizationoccurs is at least about 37, 40, 42, 45, 50, 55, 60, 65, 70, 75, or 80°C. In some embodiments, the defined temperature at which specifichybridization occurs is, or is about, 37, 40, 42, 45, 50, 55, 60, 65,70, 75, or 80° C.

A “template” refers to a polynucleotide sequence that comprises thepolynucleotide sequence to be amplified, flanked by a pair of primerhybridization sites, or adjacent to a primer hybridization site. Thus, a“target template” or “target polynucleotide sequence” comprises thetarget polynucleotide sequence adjacent to at least one primerhybridization site. In some cases, a “target template” comprises thetarget polynucleotide sequence flanked by a hybridization site for a“forward” primer and a “reverse” primer.

A “target genomic region” refers to a region of the genome of anorganism that is targeted for genome editing by a site-specific genomeediting reagent. The target genomic region can be amplified byhybridization and extension of one or more amplification primers. Thetarget genomic region can contain one or more sub-regions that contain aspecific targeted cleavage or nick site.

A “site-specific genome editing reagent” refers to a component or set ofcomponents that can be used for site-specific genome editing. Generally,such a reagent contains a targeting module and a nuclease or nickasemodule. Exemplary targeting modules contain nucleic acids, e.g., guideRNAs, such as those utilized in CRISPR/Cas-type systems. Alternatively,the targeting module can be, or be derived from, a transcription factordomain, or a TAL effector DNA binding domain. For example, a zinc-fingerdomain can be employed as a targeting moiety. Exemplary nuclease ornickase modules include, but are not limited to a type IIS restrictionendonuclease (e.g., FokI), a Cas nuclease (e.g., Cas9), or a derivativethereof. In some cases, the site-specific genome editing reagentutilizes a combination of a guide RNA, a “dead” Cas nuclease, and a typeIIS restriction endonuclease. Other variations are known in the art.

As used herein, “nucleic acid” means DNA, RNA, single-stranded,double-stranded, or more highly aggregated hybridization motifs, and anychemical modifications thereof. Modifications include, but are notlimited to, those providing chemical groups that incorporate additionalcharge, polarizability, hydrogen bonding, electrostatic interaction,points of attachment and functionality to the nucleic acid ligand basesor to the nucleic acid ligand as a whole. Such modifications include,but are not limited to, peptide nucleic acids (PNAs), phosphodiestergroup modifications (e.g., phosphorothioates, methylphosphonates),2′-position sugar modifications, 5-position pyrimidine modifications,8-position purine modifications, modifications at exocyclic amines,substitution of 4-thiouridine, substitution of 5-bromo or 5-iodo-uracil;backbone modifications, methylations, unusual base-pairing combinationssuch as the isobases, isocytidine and isoguanidine and the like. Nucleicacids can also include non-natural bases, such as, for example,nitroindole. Modifications can also include 3′ and 5′ modificationsincluding but not limited to capping with a fluorophore (e.g., quantumdot) or another moiety.

A “polymerase” refers to an enzyme that performs template-directedsynthesis of polynucleotides, e.g., DNA and/or RNA. The term encompassesboth the full length polypeptide and a domain that has polymeraseactivity. DNA polymerases are well-known to those skilled in the art,including but not limited to DNA polymerases isolated or derived fromPyrococcus furiosus, Thermococcus litoralis, and Thermotoga maritime, ormodified versions thereof. Additional examples of commercially availablepolymerase enzymes include, but are not limited to: Klenow fragment (NewEngland Biolabs® Inc.), Taq DNA polymerase (QIAGEN), 9° N™ DNApolymerase (New England Biolabs® Inc.), Deep Vent™ DNA polymerase (NewEngland Biolabs® Inc.), Manta DNA polymerase (Enzymatics®), Bst DNApolymerase (New England Biolabs® Inc.), and phi29 DNA polymerase (NewEngland Biolabs® Inc.).

Polymerases include both DNA-dependent polymerases and RNA-dependentpolymerases such as reverse transcriptase. At least five families ofDNA-dependent DNA polymerases are known, although most fall intofamilies A, B and C. Other types of DNA polymerases include phagepolymerases. Similarly, RNA polymerases typically include eukaryotic RNApolymerases I, II, and III, and bacterial RNA polymerases as well asphage and viral polymerases. RNA polymerases can be DNA-dependent andRNA-dependent.

As used herein, the term “partitioning” or “partitioned” refers toseparating a sample into a plurality of portions, or “partitions.”Partitions are generally physical, such that a sample in one partitiondoes not, or does not substantially, mix with a sample in an adjacentpartition. Partitions can be solid or fluid. In some embodiments, apartition is a solid partition, e.g., a microchannel. In someembodiments, a partition is a fluid partition, e.g., a droplet. In someembodiments, a fluid partition (e.g., a droplet) is a mixture ofimmiscible fluids (e.g., water and oil). In some embodiments, a fluidpartition (e.g., a droplet) is an aqueous droplet that is surrounded byan immiscible carrier fluid (e.g., oil).

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 depicts an exemplary schematic for simultaneous detection ofhomology dependent repair (HDR) and non-homologous end joining (NHEJ)mutations in a digital amplification (e.g., digital PCR (dPCR)) assay.Forward and reverse amplification primers flank a genomic regiontargeted by a genome editing reagent. An amplicon (e.g., approximately100-400 bp in length, or longer) is generated by polymerase chainreaction or other amplification methods. A reference probe hybridizesto, and detects, all amplicon alleles (WT, NHEJ, HDR). An HDR probedetects the HDR edit site. A non-extendible HDR dark probe blockscross-reactivity to wild-type. An HDR dark probe is not alwaysnecessary. An NHEJ drop-off probe hybridizes to the wild-type cut site.NHEJ mutations at the cut site result in loss of hybridization of theNHEJ drop-off probe. In some cases, the NHEJ drop-off probe canhybridize to a region overlapping the target site of the HDR probe andthus block potential HDR cross-reactivity with wild-type template. Theposition of the NHEJ drop-off probe can vary by cutting strategy; it maylie adjacent to the edit site and not overlap the HDR site at all.

FIG. 2 illustrates how the specific placement of probes for the digitalamplification assay can vary based on the specific cutting strategyused. NHEJ drop-off probes can be designed to hybridize to the site ofDNA cutting. For example, in CRISPR-Cas9, one double-strand cut is made3 bp upstream of the PAM site (NGG) where the guide RNA (gRNA) istargeted (SEQ ID NO:3). The NHEJ drop-off probe can be designed totarget, and thus interrogate, this cut site. The HDR event might betargeted up to 50-100 bp away. The HDR probe can thus be targeted tohybridize at a certain distance from the NHEJ drop-off probe based onguide and donor sequences used. For Cas9-Nickase, two NHEJ sites arecreated by using paired gRNAs to cause a single strand break at eachgRNA target site (SEQ ID NOS:1-2). In this case, the dPCR assay couldcontain two NHEJ drop-off probes. This strategy is generalizable toother non-CRISPR cut or nick strategies, like those utilizing Taleeffector nucleases or nickases (TALENs) and Zinc-Finger Nucleases ornickases (ZFNs).

FIG. 3 illustrates several variations on the NHEJ and HDR quantificationassay strategy. The choice of assay design can be based on the type ofgenome editing reagent employed, which determines the type and locationof expected HDR and NHEJ modifications. An HDR dark probe (not shown),can also be utilized to increase the stringency of the HDR probe. Thegenome editing reagent cut-site is depicted by an X, which alsocorresponds to the location of subsequent NHEJ-induced mutations. Thisfigure illustrates assay design for various CRISPR-Cas9 genome editingstrategies, but this approach can be applied to TALENs and ZFNs.

FIG. 4 illustrates an “all-mutation assay” that can be utilized todetect NHEJ and HDR mutations when several different genome editingreagents are employed (e.g., CRISPR/Cas, CRISPR/Cas9-Nickase,dCas9-FokI, etc.). In this example, one primer pair, one referenceprobe, one HDR probe and one HDR-dark probe (not shown) is used, alongwith a total of three NHEJ drop-off probes, each one positioned at agiven predicted cut site (depicted by an X) as dictated by the threedifferent genome editing reagents. A reduction of signal from any one ofthe detectably labeled NHEJ drop-off probes indicates an NHEJ event atany of the three possible cut sites generated by the genome editingreagents. Loss of signal from more than one NHEJ drop-off probe due tomultiple NHEJ events or one extended event will result in an increase inthe magnitude of signal loss.

FIG. 5 illustrates an embodiment in which long probes (e.g., greaterthan 50 bp) are utilized. The NHEJ drop-off probe (which hybridizes tothe NHEJ sequence) can detect NHEJ mutations along an extended region ofthe amplicon. In some cases, the NHEJ drop-off probe can target a sitethat overlaps the hybridization site of the HDR probe and serve toincrease the stringency of the HDR probe. Alternatively, an HDR darkprobe can be used (not shown).

FIG. 6 illustrates an example assay design to quantify NHEJ (SEQ IDNOS:9, 15 and 16) and HDR (SEQ ID NO:10) mutations at the genomic siteRBM20 R636S (SEQ IDS NOS:12, 32, 33-13, 34 and 35, respectively, inorder of appearance) with three different genome editing reagents(CRISPR-Cas9, Cas9-Nickase, and dCas9-FokI).

FIG. 7 illustrates data resulting from a simulated assay depicted inFIG. 6 with a CRISPR-Cas9 genome editing reagent. The results aregenerated using wild-type only genomic DNA as input.

FIG. 8 illustrates data resulting from a simulated assay depicted inFIG. 6 with a CRISPR-Cas9 genome editing reagent. The results aregenerated by doping a sample of wild-type template at a concentration of0.5 copies per droplet with 5% simulated HDR and 5% simulated NHEJtemplate. The data can be analyzed to determine the concentration ofedited genomes in a sample. Such data can be used to determine the HDR,NHEJ, or both HDR and NHEJ genome editing efficiency of a genome editingreagent or protocol.

FIG. 9A-B illustrates a scheme for targeting the GRN locus forintroduction of a point mutation (SEQ ID NOS:20-21). A pair of guideRNAs (gRNAs) for Cas9 nickase were designed to localize a Cas9 nickasein two proximal positions in the GRN locus (SEQ ID NOS:17-18) togenerate a pair of proximal single stranded nicks on opposite strands ofthe target locus. A donor oligonucleotide (SEQ ID NO: 19) encoding thedesired mutation can act as a template during homology directed repair(HDR) of the lesion. FIG. 9B depicts an alignment of two sequences (SEQID NOS:22-24) from an isolated iPS cell clone edited by the genomeediting reagent depicted in FIG. 9A. The clone has the C>T HDR mutationin one allele (SEQ ID NO:23) and a 24-bp deletion in the other allele(SEQ ID NO:24). These results indicate that a combination of HDR andNHEJ repair can happen in a single cell, providing compound heterozygousmutant cells.

FIG. 10A-B illustrates a scheme for targeting the RBM20 locus (SEQ IDNOS:25-26 and 28-29) for introduction and detection of an HDR mutation(SEQ ID NOS:30-31). The editing reagents, donor oligonucleotide (SEQ IDNO:27), and a TaqMan based PCR assay detection scheme are depicted inFIG. 10A. Exemplary Cas9, Cas9-nickase, and TALEN genome editingreagents are further depicted in FIG. 10B.

FIG. 11A-C depicts a ddPCR assay for detection of HDR mutations. FIG.11A depicts the basics of the ddPCR assay. FIG. 11B depicts atwo-dimensional plot of the resulting ddPCR data for a 1:1 mixture oftarget genomic regions containing wild-type and HDR alleles. FIG. 11Cdepicts ddPCR data for a dilution series of the HDR allele in awild-type background.

FIG. 12 depicts a scheme for a ddPCR assay to simultaneously detect bothHDR and NHEJ mutations.

FIG. 13A-D depict data from validation of the assay depicted in FIG. 12.Genomic DNA from WT HEK293 cells without FIG. 13A and with FIG. 13B 5%gBlock DNA of HDR and NHEJ alleles were analyzed by the Cas9 probemixture shown in FIG. 4 (Ref, HDR, NHEJ, and Dark probes). FIG. 13Cillustrates the quantitative performance of the assay with differentratios of wild-type and mutant target genomic regions. FIG. 13D depictsdata from HEK293 cells treated with a Cas9 genome editing reagent, asshown in FIG. 10A. Both HDR+ and NHEJ+ populations were observed in thisassay.

FIG. 14 depicts a comparison of the HDR and NHEJ mutation generatingactivities of various genome editing strategies. HEK293 cells or humaniPS cells were transfected with RBM20 TALENs, Cas9, or Cas9 nickasestogether with an RBM20 R636S single strand DNA oligonucleotide donor tointroduce the C>A mutation as shown in FIG. 10A.

DETAILED DESCRIPTION OF THE INVENTION I. Introduction

Reagents for site-specific genome editing are becoming increasinglyadvanced. Generally, such reagents target a genomic region and induce adouble stranded cut or two single stranded nicks into the DNA within thetarget region. Repair of the cutting or nicking can proceed via twoalternative pathways. In non-homologous end joining (NHEJ), the cut ornicked ends of a DNA strand are directly ligated without the need for ahomologous template nucleic acid. NHEJ can lead to the addition, thedeletion, substitution, or a combination thereof, of one or morenucleotides at the repair site. In homology directed repair, the cut ornicked ends of a DNA strand are repaired by polymerization from ahomologous template nucleic acid. Thus, the original sequence isreplaced with the sequence of the template. The homologous templatenucleic acid can be provided by homologous sequences elsewhere in thegenome (sister chromatids, homologous chromosomes, or repeated regionson the same or different chromosomes). Alternatively, an exogenoustemplate nucleic acid can be introduced to obtain a specific HDRmutation.

Although current genome editing reagents can be quite specific, theintroduction of off-target mutations can be a concern. Such off-targetmutations can be difficult to identify, as they can occur throughout thegenome. Several methods can be used to reduce the frequency ofoff-target mutations. For example, a low concentration or activity ofgenome editing reagent can be used to reduce the overall frequency ofmutation. By reducing both on-target and off-target mutation frequency,the chances that a single cell will contain both on-target andoff-target mutations is reduced. However, this makes it more difficultto identify the desired edited cells in a background population ofun-edited cells.

Alternatively, genome editing reagents that require multiple targetrecognition events can be utilized. For example, a genome editingreagent can be designed that includes a pair of nickases, where eachnickase is directed to and nicks DNA at a proximal location within atarget genomic region. Off-target activity from individual nickases ofthe pair is overwhelmingly likely to result in a single nick at theoff-target site, which is quickly repaired by the high-fidelitybase-excision repair pathway. Such nickase pairs can readily begenerated by a variety of methods. For example, a cell can be contactedwith a Cas9 nickase mutant, such as the D10A or H840A variants, and apair of guide RNAs directed to proximal sites within the target genomicregion. See, Shen et al., Nat Methods. 2014 Apr;11(4):399-402.

As another example, a genome editing reagent containing an obligateheterodimer nuclease can be used to reduce off-target mutations. Such agenome editing reagent can be designed to only generate double strandedbreaks when obligate heterodimer nucleases are formed at the targetgenomic region by site-specific recruitment of each monomer component toan adjacent target half-site. Exemplary obligate heterodimer nucleasesinclude, but are not limited to, those described in U.S. patentapplication Ser. No. 13/812,857. The targeting function can be providedby a nuclease defective Cas9 (dCas9) and appropriate guide RNAs, a pairof TALENs, or any other nucleic acid sequence specific targeting method.

Despite these and other recent advances in the development ofsite-specific genome editing reagents, it can be useful to quantify theamount of genome editing achieved in a sample. Such quantification canbe useful, in some cases, for optimizing genome editing conditions, orenriching a population of edited cells by sib-selection techniques. Forexample, genome editing conditions can be optimized to decrease, orincrease, the type or amount of NHEJ mutation in comparison to HDRmutation. As another example, genome editing conditions can be optimizedto increase the efficiency of editing, thus allowing the use of a lowconcentration or activity of genome editing reagent without undulyreducing the amount of editing achieved.

Miyaoka et al. Nat Methods. 2014 March; 11(3):291-3, describe a methodof quantifying HDR mutations by digital PCR and applying sib-selectionto enrich for genome edited cells. This method relies on the use ofsequence specific probes for detection of a predetermined mutationintroduced by a template nucleic acid during homology directed repair ofDNA damage. However, probes for direct detection of NHEJ mutationscannot be so-designed because the type (e.g., insertion or deletion) andextent (e.g., number of base pairs) of the mutation cannot be predicted.In some cases, depending on the genome editing reagent employed, theprecise location of the NHEJ mutation also cannot be predicted.

Described herein are methods for quantification of NHEJ mutations. Alsodescribed herein are methods for simultaneous quantification of NHEJ andHDR mutations.

II. Methods

Described herein are methods for detecting or quantifying homologydirected repair (HDR) genome editing products, non-homologous endjoining (NHEJ) genome editing products, or a combination thereof in asample by digital amplification. Generally the sample is a sample ofcells, a sample of genomes extracted from a sample of cells, orfragments thereof. The methods can be used to determine a degree ofgenome editing achieved for a sample, for identifying optimal conditionsfor genome editing, or to guide enrichment of populations of cells forgenome editing products (e.g., by sib-selection).

The cells, or genomes extracted from cells, can be contacted with asite-specific genome editing reagent under conditions suitable for thegenome editing of a target genomic region. In some cases, the cells orgenomes are contacted with an HDR template nucleic acid to introduce apre-determined HDR mutation into the genome. After contact with asite-specific genome editing reagent, the degree of genome editingachieved can be determined using one or more methods described herein.The genome editing reagent can contain one or more nucleases ornickases, or a combination thereof.

A. Detection of NHEJ

In some embodiments, the methods include detecting or quantifying NHEJgenome editing products in a sample. The NHEJ genome editing productscan be detected or quantified using a reference probe and an NHEJdrop-off probe. The methods for detecting or quantifying NHEJ genomeediting products in a sample can include providing a sample of nucleicacid from the cells or genomes contacted with a genome editing reagent,and forming a reaction mixture containing a reference probe and an NHEJdrop-off probe. The sample can contain NHEJ mutations, HDR mutations, ora combination thereof.

The reaction mixture can be partitioned to form a plurality of mixturepartitions having an average volume, the mixture partitions containing:i) a target genomic region; ii) a DNA dependent DNA polymerase; iii) aforward and a reverse oligonucleotide amplification primer, wherein theforward and reverse primers hybridize to opposite strands of, and flank,the target genomic region, and wherein the primers are configured toamplify the target genomic region in the presence of the polymerase; andiv) a detectably labeled oligonucleotide reference probe that hybridizesto the target genomic region, regardless of the allele (i.e., hybridizesto wild-type, HDR, and NHEJ edited target genomic regions).

The mixture partitions can further contain one or more NHEJ drop-offprobes. Generally, the NHEJ drop-off probes are designed to hybridize toa sub-region of the target genomic region containing a cut or nick sitetargeted by the site-specific genome editing reagent. The design of theone or more NHEJ drop-off probes can vary with the type of genomeediting reagent to which the sample has been contacted.

For example, if the genome editing reagent is a Cas9 nuclease and aguide RNA, cut sites are generally 3-5 base pairs directly upstream of aprotospacer adjacent motif (PAM). The PAM generally consists of thesequence NGG, although some other PAM sequences can be utilized, such asNGA or NAG. Thus, cut sites can be, for instance, either [5′-20nttarget-NGG-3′] or [5′-CCN-20nt target-3′], as it is equally efficaciousto target the coding or non-coding strand of DNA. When the target siteis 5′-20nt target-NGG, the predicted cut-site is approximately 3-5 basepairs upstream of the 5′ end of the NGG PAM. In such cases, the NHEJdrop-off probe can be designed to hybridize to a sub-region containingthis predicted cut-site.

As another example, the genome editing reagent can be a pair of guideRNAs targeted to sites adjacent to PAM sequences on opposite strands ofthe target genomic region, each guide RNA complexed with a nucleasedefective, or dead, Cas9 nuclease (dCas9) that is fused to monomer of anobligate heterodimer of a type IIS restriction nuclease (e.g., FokI). Insuch cases, the cut site is generally from 12 to 21 base pairs betweenthe adjacent PAM sequences on the opposite strands of the targetedgenomic region. Thus, the NHEJ drop-off probe can be designed tohybridize to a sub-region containing a predicted cut site from 12 to 21base pairs between the adjacent PAM sequences. Similar rules can beutilized to design NHEJ drop-off probes for other genome editingreagents.

In some cases, the range of locations of the predicted cut-site can belarger than 12-21 base pairs. In such cases, multiple NHEJ drop offprobes can be utilized to cover a larger area of potential NHEJmutations. Alternatively, the NHEJ drop-off probes can be increased inlength; however, in some cases, increasing the length of the drop-offprobe can decrease the ability to detect single nucleotide genome edits.In some cases, the genome editing reagent is designed to create multiplecuts or nicks. In such cases, multiple NHEJ drop off probes can bedesigned to hybridize to the multiple cuts or nicks. For example, thegenome editing reagent can be a pair of nickases targeted to nick thetarget genomic region in proximal locations (e.g., a pair of nick sitesseparated by less than 1 kb, less than 500 bp, less than 250 bp, lessthan 200 bp, less than 150 bp, or less than 100 bp) and on oppositestrands of the target genomic region. In this example, a pair of NHEJdrop-off probes can be designed to detect NHEJ mutations at each of thenick sites.

The NHEJ-drop off probes can be from 10 to 35 nucleotides in length. Insome cases, the NHEJ-drop off probes are 12 to 30 nucleotides, or 18 to30 nucleotides in length. In some cases, the NHEJ-drop off probes are12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29,or 30 nucleotides in length. Generally, the probes are designed tospecifically hybridize to the wild-type target sequence but not tohybridize when a mutation is present in the target sequence under theamplification and/or detection conditions of the assay. This specificityof hybridization can be achieved by altering the length of the probe,the GC content, or the amplification and/or detection conditions (e.g.,temperature, salt content, etc.).

In some cases, the NHEJ drop-off probe is sensitive to (i.e., detects)both HDR mutations and NHEJ mutations. For example, the genome editingreagent can include an exogenous HDR template nucleic acid. The templatenucleic acid can be used as a template to repair a region encompassing,or within, the double strand breaks or paired nicks introduced by thegenome editing reagent. Thus, any mutations present in the HDR templatenucleic acid relative to the wild-type genome will be introduced. Whenthe HDR site is proximal to, or at, the target cut site, the NHEJdrop-off probe can hybridize to both potential NHEJ edit sites and thepotential HDR edit site. In such cases, the NHEJ drop-off probe candetect both HDR mutations and NHEJ mutations by failing to hybridize totarget genomic regions containing such mutations.

Moreover, NHEJ mutations and HDR mutations can be distinguished by useof an HDR probe in the reaction mixture. For example, an HDR probe asdescribed herein or an HDR probe as described in Miyaoka et al. 2014.Thus, if the NHEJ drop off probe detects a mutation (HDR or NHEJ), andthe HDR probe does not, then the mutation can be classified as NHEJ.Conversely, if the NHEJ probe detects a mutation and the HDR probe alsodetects a mutation, then the mutation can be classified as HDR.Similarly, in a partition that contains multiple target genomic regions,each with a possible NHEJ and/or HDR mutation, the presence or absenceof NHEJ or HDR mutations can be determined by the relative intensity ofsignal from the different probes. Thus, for example, if a partitioncontains one target genomic region with an NHEJ mutation and one targetgenomic region with an HDR mutation, the reference probe signal willindicate multiple target genomic regions, the HDR probe signal willindicate one HDR mutated target genomic region, and the NHEJ drop offprobe signal will indicate that the multiple target genomic regionscontain a mutation.

The target genomic regions in the plurality of mixture partitions can beamplified by, e.g., subjecting the mixture partitions to PCRamplification conditions. The conditions can include a two or three stepthermal cycling protocol. In some cases, a three step thermal cyclingprotocol is utilized to ensure complete amplification of target genomicregions. For example, in some cases, if a target genomic region,including predicted cut site locations HDR mutation sites (ifapplicable) and reference probe hybridization sites, is greater thanabout 200 bp, or greater than about 400 bp, a three step amplificationprotocol may be selected.

The quantity of empty mixture partitions can be determined by detectingthe number of mixture partitions in which the reference probe, or NHEJdrop-off and reference probes, do not detect any target genomic regionamplicons. This quantity of empty mixture partitions can be referred toas N_(neg). The quantity of mixture partitions containing only NHEJedited target genomic region amplicons (N_(NHEJ)) in a sample in whichan HDR template nucleic acid was not utilized to induce HDR mutationscan be determined by detecting the number of mixture partitions in whichthe reference probe detects target genomic region amplicons, but theNHEJ drop-off probe does not detect any wild-type target genomic regionamplicons. The number of mixture partitions containing only NHEJ editedtarget genomic region amplicons can added to the number of empty mixturepartitions to obtain a value referred to as N_(total). The concentrationof NHEJ edited genomes in the sample can then be determined bycalculating a reciprocal of the average volume of the plurality ofmixture partitions multiplied by a negative natural log of the ratio ofN_(neg) divided by N_(total).

Similarly, in a reaction mixture in which HDR mutations may be present,HDR and NHEJ drop off probes can be used in combination to determine thequantity of mixture partitions containing only NHEJ edited targetgenomic region amplicons (N_(NHEJ)). For example, the quantity ofmixture partitions containing only NHEJ edited target genomic regionamplicons (N_(NHEJ)) in a sample in which an HDR template nucleic acidwas utilized to induce HDR mutations, can be determined by detecting thenumber of mixture partitions in which the reference probe detects targetgenomic region amplicons, the NHEJ drop-off probe does not detect anywild-type target genomic region amplicons, and an HDR probe does notdetect an HDR allele.

The concentration of wild-type genomes in the sample can be readilyderived from the concentration of NHEJ edited genomes in the sample bysubtracting the concentration of NHEJ edited genomes from the totalconcentration of genomes in the NHEJ reaction mixture. Alternatively,the concentration of wild-type genomes can be derived directly by addingtogether the number of: a) empty mixture partitions (N_(empty)); b)mixture partitions containing only NHEJ edited target genomic amplicons(N_(NHEJ)); and c) mixture partitions containing at least somedetectable wild-type target genomic amplicons (N_(NHEJ+WT) and N_(WT))to obtain N_(total). A value for N_(neg) can be obtained by addingtogether the number of empty mixture partitions (N_(empty)) and mixturepartitions containing only NHEJ edited target genomic amplicons(N_(NHEJ)). The concentration of wild-type genomes in the sample canthen be determined by calculating a reciprocal of the average volume ofthe plurality of mixture partitions multiplied by a negative natural logof the ratio of N_(neg) divided by N_(total).

B. Detection of NHEJ and HDR in Different Reaction Mixtures

In some embodiments, the methods include detecting or quantifying HDRgenome editing products in one reaction mixture and detecting orquantifying NHEJ genome editing products in a different reactionmixture. For example, a sample can be divided into two differentreaction mixtures and HDR and NHEJ quantified in the different reactionmixtures (i.e., an HDR reaction mixture and an NHEJ reaction mixture).The NHEJ genome editing products can be detected or quantified using areference probe and an NHEJ drop-off probe as described above. The HDRgenome editing products can be detected or quantified using a referenceprobe an HDR probe, and optionally an HDR dark probe.

The NHEJ and HDR reaction mixtures can be separately partitioned to forma plurality of mixture partitions having an average volume, the mixturepartitions containing: i) a target genomic region; ii) a DNA dependentDNA polymerase; iii) a forward and a reverse oligonucleotideamplification primer, wherein the forward and reverse primers hybridizeto opposite strands of, and flank, the target genomic region, andwherein the primers are configured to amplify the target genomic regionin the presence of the polymerase; and iv) a detectably labeledoligonucleotide reference probe that hybridizes to the target genomicregion, regardless of the allele (i.e., hybridizes to wild-type, HDR,and NHEJ edited target genomic regions).

The mixture partitions generated by partitioning the HDR reactionmixture can further contain one or more HDR probes. Generally, the HDRprobes are designed to hybridize to a sub-region of the target genomicregion containing an HDR mutation, but not to hybridize to a sub-regionthat does not contain the HDR mutation. In some cases, the HDR probe canalso detect NHEJ mutations. For example, the HDR probe can hybridize toa region overlapping the predicted cut site of the site-specific genomeediting reagent, and thus detect the presence or absence of NHEJ inducederrors at the repaired cut site. The design of the one or more HDRprobes can vary with the number and character of the mutations presenton the HDR template nucleic acid.

The HDR probes can be from 10 to 35 nucleotides in length, or longer. Insome cases, the HDR probes are 12 to 30 nucleotides, or 18 to 30nucleotides in length. In some cases, the HDR probes are 12, 13, 14, 15,16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30nucleotides in length. Generally, the probes are designed tospecifically hybridize to the HDR edited target genomic sequence but notto the wild-type target sequence under the amplification and/ordetection conditions of the assay. This specificity of hybridization canbe achieved by altering the length of the probe, the GC content, or theamplification and/or detection conditions (e.g., temperature, saltcontent, etc.).

The HDR probe can contain the same detectable label as the referenceprobe. In such cases, the signal from the HDR probe and the referenceprobe can be additive, when HDR mutated target genomic regions aredetected. Mixture partitions containing both HDR-mutated target genomicregions and non-HDR-mutated target genomic regions (e.g., wild-type) canthen be detected as a separate, but possibly overlapping, clustersituated between wild-type only and HDR only target genomic regioncontaining mixture partitions.

The HDR mixture partitions can contain an HDR dark probe. The HDR darkprobe can increase the stringency of the assay by decreasing erroneoussignal provided by binding of the HDR probe to the wild-type targetgenomic region. The HDR dark probe is generally designed to compete withthe HDR probe for binding to the wild-type target genomic region, butnot the HDR mutated target genomic region. In some cases, the HDR darkprobe and the HDR probe are the same sequence except at the nucleotidesaltered by the HDR mutation. Typically, the HDR dark probe is designedto contain a non-extendible 3′ end. An exemplary non-extendible 3′ endincludes, but is not limited to a 3′ terminal phosphate. Alternativenon-extendible 3′ ends include, but not limited to, those disclosed in,e.g., international patent application publication No. WO 2013/026027.

The target genomic regions in the plurality of HDR mixture partitionscan be amplified by, e.g., subjecting the mixture partitions to PCRamplification conditions. The conditions can include a two or three stepthermal cycling protocol. In some cases, a three step thermal cyclingprotocol is utilized to ensure complete amplification of target genomicregions. For example, in some cases, if a target genomic region,including predicted cut site locations, HDR mutation sites, andreference probe hybridization sites, is greater than about 200 bp or 400bp, a three step amplification protocol may be selected.

NHEJ and wild-type quantification can be performed by analyzing the NHEJmixture partitions as described above. The concentration of HDR editedgenomes can be derived by adding together the number of: a) emptymixture partitions (N_(empty)); b) mixture partitions containing onlyNHEJ edited target genomic amplicons (N_(NHEJ)); and c) mixturepartitions containing at least some detectable wild-type target genomicamplicons (N_(NHEJ+WT) and N_(WT)) to obtain N_(neg). A value forN_(Total) can be obtained by adding N_(neg) and mixture partitionscontaining at least some detectable HDR edited target genomic amplicons(N_(HDR), N_(HDR+WT), N_(HDR+NHEJ), and N_(HDR+NHEJ+WT)). Theconcentration of wild-type genomes in the sample can then be determinedby calculating a reciprocal of the average volume of the plurality ofmixture partitions multiplied by a negative natural log of the ratio ofN_(neg) divided by N_(total).

C. Simultaneous Detection of NHEJ and HDR

In some embodiments, the methods include simultaneously detecting orquantifying HDR and NHEJ genome editing products in a sample. The HDRand NHEJ genome editing products can be detected or quantified using areference probe as described herein, one or more NHEJ drop-off probes asdescribed herein, an HDR probe as described herein, and optionally anHDR dark probe as described herein. The methods for detecting orquantifying HDR and NHEJ genome editing products in a sample can includeproviding a sample of nucleic acid from the cells or genomes contactedwith a genome editing reagent, and forming a reaction mixture containinga reference probe, one or more NHEJ drop-off probes, an HDR probe, andoptionally an HDR dark probe. In some cases, the simultaneous detectionof NHEJ and HDR is performed by forming a single reaction mixturecontaining a reference probe, one or more NHEJ drop-off probes, an HDRprobe, and optionally an HDR dark probe. In some cases, the use of asingle reaction mixture can reduce the likelihood of user error orincrease the precision or accuracy of the assay.

The reaction mixture can be partitioned to form a plurality of mixturepartitions having an average volume, the mixture partitions containing:i) a target genomic region; ii) a DNA dependent DNA polymerase; iii) aforward and a reverse oligonucleotide amplification primer, wherein theforward and reverse primers hybridize to opposite strands of, and flank,the target genomic region, and wherein the primers are configured toamplify the target genomic region in the presence of the polymerase; andiv) a detectably labeled oligonucleotide reference probe that hybridizesto the target genomic region, regardless of the allele (i.e., hybridizesto wild-type, HDR, and NHEJ edited target genomic regions).

The mixture partitions can further contain one or more NHEJ drop-offprobes, an HDR probe, and optionally an HDR dark probe. In some cases,one of the one or more NHEJ drop-off probes hybridizes to a sub-regionof the target genomic region that overlaps with the hybridization siteof the HDR probe. In such cases, the NHEJ probe can substitute for, andrender unnecessary, the use of an HDR dark probe.

In some cases, the mixture partitions are formed in single well. In somecases, the use of a single well can reduce the likelihood of user erroror increase the precision or accuracy of the assay. For example, aplurality of droplet mixture partitions can be formed in a single well.The mixture partitions in the well can contain: i) a target genomicregion; ii) a DNA dependent DNA polymerase; iii) a forward and a reverseoligonucleotide amplification primer, wherein the forward and reverseprimers hybridize to opposite strands of, and flank, the target genomicregion, and wherein the primers are configured to amplify the targetgenomic region in the presence of the polymerase; and iv) a detectablylabeled oligonucleotide reference probe that hybridizes to the targetgenomic region, regardless of the allele (i.e., hybridizes to wild-type,HDR, and NHEJ edited target genomic regions). The mixture partitions inthe well can further contain one or more NHEJ drop-off probes, an HDRprobe, and optionally an HDR dark probe

The target genomic regions in the plurality mixture partitions can beamplified by, e.g., subjecting the mixture partitions to PCRamplification conditions. The conditions can include a two or three stepthermal cycling protocol. In some cases, a three step thermal cyclingprotocol is utilized to ensure complete amplification of target genomicregions. For example, in some cases, if a target genomic region,including predicted cut site locations, HDR mutation sites, andreference probe hybridization sites, is greater than about 200 bp, orgreater than about 400 bp, a three step amplification protocol may beselected.

NHEJ and wild-type quantification can be performed by analyzing themixture partitions as described above. The concentration of HDR editedgenomes can be derived by adding together the number of: a) emptymixture partitions (N_(empty)); b) mixture partitions containing onlyNHEJ edited target genomic amplicons (N_(NHEJ)); and c) mixturepartitions containing at least some detectable wild-type target genomicamplicons (N_(NHEJ+WT) and N_(WT)) to obtain N_(neg). A value forN_(Total) can be obtained by adding N_(neg) and mixture partitionscontaining at least some detectable HDR edited target genomic amplicons(N_(HDR), N_(HDR+WT), N_(HDR+NHEJ), and N_(HDR+NHEJ+WT)). Theconcentration of wild-type genomes in the sample can then be determinedby calculating a reciprocal of the average volume of the plurality ofmixture partitions multiplied by a negative natural log of the ratio ofN_(neg) divided by N_(total).

An exemplary schematic for simultaneous detection of HDR and NHEJ isdepicted in FIG. 1. As shown in this figure, forward and reverseamplification primers flank a genomic region targeted by a genomeediting reagent. An amplicon is generated during multiple rounds ofhybridization and extension of the amplification primers by a DNAdependent DNA polymerase (e.g., PCR). The amplicon can be approximately100-400 bp in length, or longer. Generally, the length of the ampliconis chosen to ensure that there is enough room so that all probes (e.g.,reference, NHEJ, and HDR) can bind to their target sequence in theamplicon. In some cases, it is desirable to minimize the size of theamplicon to decrease the amplification time and thereby increase thespeed of the assay. In some cases, the size of the amplicon can bedetermined by the size of the flanking regions upstream and downstreamof the target cut or nick site encompassed by the amplification primers.The flanking regions can be approximately 200 bp as depicted in FIG. 1.Alternatively the flanking regions can be approximately 50, 60, 70, 80,90, 100, 125, 150, 175, 200, 225, 250, or longer. In some cases, theupstream and downstream flanking regions are the same size. In somecase, the upstream and downstream flanking regions are different sizes.

As shown in FIG. 1, a reference probe hybridizes to, and detects, allamplicon alleles. An HDR probe detects the HDR edit site. An optionalnon-extendible HDR dark probe blocks cross-reactivity to wild-type. AnNHEJ drop-off probe hybridizes to the wild-type cut site. NHEJ (or HDR)mutations at the cut site result in loss of hybridization of the NHEJdrop-off probe. In some cases, the NHEJ drop-off probe can hybridize toa region overlapping the target site of the HDR probe and thus blockpotential HDR cross-reactivity with wild-type template, replacing thefunction of the HDR dark probe. The position of the NHEJ drop-off probecan vary by cutting strategy; it may lie adjacent to the edit site andnot overlap the HDR site at all.

An alternative exemplary schematic for simultaneous detection of HDR andNHEJ is depicted in FIG. 2. As shown in this figure, the specificplacement of probes for the digital amplification assay can vary basedon the specific cutting strategy used. NHEJ drop-off probes can bedesigned to hybridize to the site of DNA cutting. For example, inCRISPR-Cas9, one double-strand cut is made 3 bp upstream of the PAM site(NGG) where the guide RNA (gRNA) gRNA is targeted. The NHEJ drop-offprobe can be designed to target, and thus interrogate, this cut site.The HDR event might be targeted up to 50-100 bp away. The HDR probe canthus be targeted to hybridize at a certain distance from the NHEJdrop-off probe based on guide and donor sequences used.

For Cas9-Nickase, two NHEJ sites are created by using paired gRNAs and amutant Cas9 that introduces single stranded cuts (nicks) into the targetDNA. By targeting two nearby (e.g., within less than about 1 kb, lessthan about 500 bp, less than about 250 bp, less than about 200 bp, lessthan about 150 bp, or less than about 100 bp, 75 bp, 50 bp, 25 bp, 15bp, or 10 bp) sequences with the pair of gRNAs, a pair of proximalsingle strand nicks is introduced. In this case, the dPCR assay couldcontain two NHEJ drop-off probes. This strategy is generalizable toother non-CRISPR cut or nick strategies, like those utilizing Taleeffector nucleases or nickases (TALENs) and Zinc-Finger Nucleases ornickases (ZFNs).

Several alternative exemplary schematics for simultaneous detection ofHDR and NHEJ are depicted in FIG. 3. The choice of assay design can bebased on the type of genome editing reagent employed, which determinesthe type and location of expected HDR and NHEJ modifications. An HDRdark probe (not shown), can also be utilized to increase the stringencyof the HDR probe. The genome editing reagent cut-site is depicted by anX, which also corresponds to the location of subsequent NHEJ-inducedmutations. This figure illustrates assay design for various CRISPR-Cas9genome editing strategies, but this approach can be applied to TALENsand ZFNs.

FIG. 4 illustrates an “all-mutation assay” that can be utilized todetect NHEJ and HDR mutations when several different genome editingreagents are employed (e.g., CRISPR/Cas, CRISPR/Cas9-Nickase,dCas9-FokI, etc.). In this example, one primer pair, one referenceprobe, one HDR probe and one HDR-dark probe (not shown) is used, alongwith a total of three NHEJ drop-off probes, each one positioned at agiven predicted cut site (depicted by an X) as dictated by the threedifferent genome editing reagents. A reduction of signal from any one ofthe detectably labeled NHEJ drop-off probes indicates an NHEJ event atany of the three possible cut sites generated by the genome editingreagents. Loss of signal from more than one NHEJ drop-off probe due tomultiple NHEJ events or one extended event will result in an increase inthe magnitude of signal loss.

FIG. 5 illustrates an embodiment in which long probes (e.g., greaterthan 50 bp) are utilized. The NHEJ drop-off probe (which hybridizes tothe NHEJ sequence) can detect NHEJ mutations along an extended region ofthe amplicon. In some cases, the NHEJ drop-off probe can target a sitethat overlaps the hybridization site of the HDR probe and serve toincrease the stringency of the HDR probe. Alternatively, an HDR darkprobe can be used (not shown).

D. Optimizing Genome Editing

One or more of the methods described herein can be used to foridentifying an optimized condition for genome editing of a cell. Forexample, site-specific genome editing can be performed on a plurality ofcells under a first set of conditions to provide first sample of cells;and a second set of conditions to provide a second sample of cells. Insome cases, editing is performed under a third, fourth, fifth, sixth,etc. number of conditions. Genomic DNA can be extracted from the cells,and the methods described above utilized to quantify a number of NHEJedited target genomic regions and/or HDR edited target genomic regionsin the different samples of cells to determine a genome editingefficiency for the different conditions. The genome editing efficiencyfor the different conditions can be compared to identify a set ofconditions that provides a higher genome editing efficiency. The higherefficiency can be selected as the optimized condition for genomeediting. In some cases, the higher efficiency of HDR editing isidentified as the optimized condition for genome editing. In some cases,the higher efficiency of NHEJ editing is identified as the optimizedcondition for genome editing. In some cases, the higher ratio of theefficiency of NHEJ to HDR editing is identified as the optimizedcondition for genome editing. In some cases, the higher ratio of theefficiency of HDR to NHEJ editing is identified as the optimizedcondition for genome editing.

In some cases, the different conditions comprise differentconcentrations of genome editing reagents. In some cases, the differentconditions comprise different types of genome editing reagents. Forinstance, a zinc-finger nuclease can be compared to a CRISPR/Casreagent, e.g., with all other variables held constant. In some cases,the different conditions comprise the same or different type of genomeediting reagent targeted to different target genomic regions. Thus, forexample, the genome editing efficiency targeted to a region of closedchromatin can be compared to the genome editing efficiency targeted to aregion of open chromatin.

E. Partitioning

Samples can be partitioned into a plurality of mixture partitions. Theuse of partitioning can be advantageous to reduce backgroundamplification, reduce amplification bias, increase throughput, provideabsolute or relative quantitative detection, or a combination thereof.Partitioning can also allow multiplex detection of different targets (ordifferent mutations in a target). Partitions can include any of a numberof types of partitions, including solid partitions (e.g., wells ortubes) or fluid partitions (e.g., aqueous droplets within an oil phase).In some embodiments, the partitions are droplets. In some embodiments,the partitions are micro channels. Methods and compositions forpartitioning a sample are described, for example, in published patentapplications WO 2010/036352, US 2010/0173394, US 2011/0092373, and US2011/0092376, the entire content of each of which is incorporated byreference herein.

In some cases, samples are partitioned and detection reagents (e.g.,probes, enzyme, etc.) are incorporated into the partitioned samples. Inother cases, samples are contacted with detection reagents (e.g.,probes, enzyme, etc.) and the sample is then partitioned. In someembodiments, reagents such as probes, primers, buffers, enzymes,substrates, nucleotides, salts, etc. are mixed together prior topartitioning, and then the sample is partitioned. In some cases, thesample is partitioned shortly after mixing reagents together so thatsubstantially all, or the majority, of reactions (e.g., DNAamplification, DNA cleavage, etc.) occur after partitioning. In othercases, the reagents are mixed at a temperature in which reactionsproceed slowly, or not at all, the sample is then partitioned, and thereaction temperature is adjusted to allow the reaction to proceed. Forexample, the reagents can be combined on ice, at less than 5° C., or at0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19,20, 20-25, 25-30, or 30-35° C. or more. In general, one of skill in theart will know how to select a temperature at which the one or morereactions are inhibited. In some cases, a combination of temperature andtime are utilized to avoid substantial reaction prior to partitioning.

Additionally, reagents and sample can be mixed using one or more hotstart enzymes, such as a hot start DNA-Dependent DNA polymerase. Thus,sample and one or more of buffers, salts, nucleotides, probes, labels,enzymes, etc. can be mixed and then partitioned. Subsequently, thereaction catalyzed by the hot start enzyme, can be initiated by heatingthe mixture partitions to activate the one or more hot-start enzymes.

Additionally, sample and reagents (e.g., one or more of buffers, salts,nucleotides, probes, labels, enzymes, etc.) can be mixed togetherwithout one or more reagents necessary to initiate an intended reaction(e.g., DNA amplification). The mixture can then be partitioned into aset of first partition mixtures and then the one or more essentialreagents can be provided by fusing the set of first partition mixtureswith a set of second partition mixtures that provide the essentialreagent. Alternatively, the essential reagent can be added to the firstpartition mixtures without forming second partition mixtures. Forexample, the essential reagent can diffuse into the set of firstpartition mixture water-in-oil droplets. As another example, the missingreagent can be directed to a set of micro channels which contain the setof first partition mixtures.

In some embodiments, the sample is partitioned into a plurality ofdroplets. In some embodiments, a droplet comprises an emulsioncomposition, i.e., a mixture of immiscible fluids (e.g., water and oil).In some embodiments, a droplet is an aqueous droplet that is surroundedby an immiscible carrier fluid (e.g., oil). In some embodiments, adroplet is an oil droplet that is surrounded by an immiscible carrierfluid (e.g., an aqueous solution). In some embodiments, the dropletsdescribed herein are relatively stable and have minimal coalescencebetween two or more droplets. In some embodiments, less than 0.0001%,0.0005%, 0.001%, 0.005%, 0.01%, 0.05%, 0.1%, 0.5%, 1%, 2%, 3%, 4%, 5%,6%, 7%, 8%, 9%, or 10% of droplets generated from a sample coalesce withother droplets. The emulsions can also have limited flocculation, aprocess by which the dispersed phase comes out of suspension in flakes.

In some embodiments, the droplet is formed by flowing an oil phasethrough an aqueous sample comprising identification signatures to bedetected. In some embodiments, the aqueous sample comprising theidentification signatures to be detected further comprises a bufferedsolution and two or more probes for detecting the identificationsignatures.

The oil phase can comprise a fluorinated base oil which can additionallybe stabilized by combination with a fluorinated surfactant such as aperfluorinated polyether. In some embodiments, the base oil comprisesone or more of a HFE 7500, FC-40, FC-43, FC-70, or another commonfluorinated oil. In some embodiments, the oil phase comprises an anionicfluorosurfactant. In some embodiments, the anionic fluorosurfactant isAmmonium Krytox (Krytox-AS), the ammonium salt of Krytox FSH, or amorpholino derivative of Krytox FSH. Krytox-AS can be present at aconcentration of about 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%,0.9%, 1.0%, 2.0%, 3.0%, or 4.0% (w/w). In some embodiments, theconcentration of Krytox-AS is about 1.8%. In some embodiments, theconcentration of Krytox-AS is about 1.62%. Morpholino derivative ofKrytox FSH can be present at a concentration of about 0.1%, 0.2%, 0.3%,0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1.0%, 2.0%, 3.0%, or 4.0% (w/w). Insome embodiments, the concentration of morpholino derivative of KrytoxFSH is about 1.8%. In some embodiments, the concentration of morpholinoderivative of Krytox FSH is about 1.62%.

In some embodiments, the oil phase further comprises an additive fortuning the oil properties, such as vapor pressure, viscosity, or surfacetension. Non-limiting examples include perfluorooctanol and1H,1H,2H,2H-Perfluorodecanol. In some embodiments,1H,1H,2H,2H-Perfluorodecanol is added to a concentration of about 0.05%,0.06%, 0.07%, 0.08%, 0.09%, 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%,0.8%, 0.9%, 1.0%, 1.25%, 1.50%, 1.75%, 2.0%, 2.25%, 2.5%, 2.75%, or 3.0%(w/w). In some embodiments, 1H,1H,2H,2H-Perfluorodecanol is added to aconcentration of about 0.18% (w/w).

In some embodiments, the emulsion is formulated to produce highlymonodisperse droplets having a liquid-like interfacial film that can beconverted by heating into microcapsules having a solid-like interfacialfilm; such microcapsules can behave as bioreactors able to retain theircontents through an incubation period. The conversion to microcapsuleform can occur upon heating. For example, such conversion can occur at atemperature of greater than about 40°, 50°, 60°, 70°, 80°, 90°, or 95°C. During the heating process, a fluid or mineral oil overlay can beused to prevent evaporation. Excess continuous phase oil can be removedprior to heating, or not. The microcapsules can be resistant tocoalescence and/or flocculation across a wide range of thermal andmechanical processing.

Following conversion, the microcapsules can be stored at about −70°,−20°, 0°, 3°, 4°, 5°, 6°, 7°, 8°, 9°, 10°, 15°, 20°, 25°, 30°, 35°, or40° C. In some embodiments, these capsules are useful for storage ortransport of partition mixtures. For example, a sample can be collectedat one location, partitioned into droplets containing enzymes, buffers,probes, and/or primers, optionally one or more amplification reactionscan be performed, the partitions can then be heated to performmicroencapsulation, and the microcapsules can be stored or transportedfor further analysis.

The microcapsule partitions can contain one or more probes (e.g.,labeled probes as described herein) and can resist coalescence,particularly at high temperatures. Accordingly, the capsules can beincubated at a very high density (e.g., number of partitions per unitvolume). In some embodiments, greater than 100,000, 500,000, 1,000,000,1,500,000, 2,000,000, 2,500,000, 5,000,000, or 10,000,000 partitions canbe incubated per mL. In some embodiments, the sample-probe incubationsoccur in a single well, e.g., a well of a microtiter plate, withoutinter-mixing between partitions. The microcapsules can also containother components necessary for the incubation.

In some embodiments, the sample is partitioned into at least 500partitions, at least 1000 partitions, at least 2000 partitions, at least3000 partitions, at least 4000 partitions, at least 5000 partitions, atleast 6000 partitions, at least 7000 partitions, at least 8000partitions, at least 10,000 partitions, at least 15,000 partitions, atleast 20,000 partitions, at least 30,000 partitions, at least 40,000partitions, at least 50,000 partitions, at least 60,000 partitions, atleast 70,000 partitions, at least 80,000 partitions, at least 90,000partitions, at least 100,000 partitions, at least 200,000 partitions, atleast 300,000 partitions, at least 400,000 partitions, at least 500,000partitions, at least 600,000 partitions, at least 700,000 partitions, atleast 800,000 partitions, at least 900,000 partitions, at least1,000,000 partitions, at least 2,000,000 partitions, at least 3,000,000partitions, at least 4,000,000 partitions, at least 5,000,000partitions, at least 10,000,000 partitions, at least 20,000,000partitions, at least 30,000,000 partitions, at least 40,000,000partitions, at least 50,000,000 partitions, at least 60,000,000partitions, at least 70,000,000 partitions, at least 80,000,000partitions, at least 90,000,000 partitions, at least 100,000,000partitions, at least 150,000,000 partitions, or at least 200,000,000partitions.

In some embodiments, the sample is partitioned into a sufficient numberof partitions such that at least a majority of partitions have no morethan 1-5 target genomic regions (e.g., no more than about 0.5, 1, 2, 3,4, or 5 target genomic regions). In some embodiments, on average about0.5, 1, 2, 3, 4, or 5 target genomic regions are present in eachpartition. In some embodiments, on average about 0.001, 0.005, 0.01,0.05, 0.1, 0.5, 1, 2, 3, 4, or 5 target genomic regions are present ineach partition. In some embodiments, at least one partition contains notarget genomic regions (the partition is “empty”). In some embodiments,at least about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, or 10% of thepartitions contain no target genomic regions. Generally, partitions cancontain an excess of enzyme, probes, and primers such that each mixturepartition is likely to successfully amplify any target genomic regionspresent in the partition.

In some embodiments, the droplets that are generated are substantiallyuniform in shape and/or size. For example, in some embodiments, thedroplets are substantially uniform in average diameter. In someembodiments, the droplets that are generated have an average diameter ofabout 0.001 microns, about 0.005 microns, about 0.01 microns, about 0.05microns, about 0.1 microns, about 0.5 microns, about 1 microns, about 5microns, about 10 microns, about 20 microns, about 30 microns, about 40microns, about 50 microns, about 60 microns, about 70 microns, about 80microns, about 90 microns, about 100 microns, about 150 microns, about200 microns, about 300 microns, about 400 microns, about 500 microns,about 600 microns, about 700 microns, about 800 microns, about 900microns, or about 1000 microns. In some embodiments, the droplets thatare generated have an average diameter of less than about 1000 microns,less than about 900 microns, less than about 800 microns, less thanabout 700 microns, less than about 600 microns, less than about 500microns, less than about 400 microns, less than about 300 microns, lessthan about 200 microns, less than about 100 microns, less than about 50microns, or less than about 25 microns. In some embodiments, thedroplets that are generated are non-uniform in shape and/or size.

In some embodiments, the droplets that are generated are substantiallyuniform in volume. For example, the standard deviation of droplet volumecan be less than about 1 picoliter, 5 picoliters, 10 picoliters, 100picoliters, 1 nL, or less than about 10 nL. In some cases, the standarddeviation of droplet volume can be less than about 10-25% of the averagedroplet volume. In some embodiments, the droplets that are generatedhave an average volume of about 0.001 nL, about 0.005 nL, about 0.01 nL,about 0.02 nL, about 0.03 nL, about 0.04 nL, about 0.05 nL, about 0.06nL, about 0.07 nL, about 0.08 nL, about 0.09 nL, about 0.1 nL, about 0.2nL, about 0.3 nL, about 0.4 nL, about 0.5 nL, about 0.6 nL, about 0.7nL, about 0.8 nL, about 0.9 nL, about 1 nL, about 1.5 nL, about 2 nL,about 2.5 nL, about 3 nL, about 3.5 nL, about 4 nL, about 4.5 nL, about5 nL, about 5.5 nL, about 6 nL, about 6.5 nL, about 7 nL, about 7.5 nL,about 8 nL, about 8.5 nL, about 9 nL, about 9.5 nL, about 10 nL, about11 nL, about 12 nL, about 13 nL, about 14 nL, about 15 nL, about 16 nL,about 17 nL, about 18 nL, about 19 nL, about 20 nL, about 25 nL, about30 nL, about 35 nL, about 40 nL, about 45 nL, or about 50 nL.

F. Monitoring of Genome Edited Cells

The future of gene editing may require monitoring of the gene edits thathave been made in the populations of genome edited cells. The monitoringcan be performed on a population of genome edited therapeutic cells thatare transfused into patients (humans or animals). The monitoring can beperformed on a population of genome edited cells that are cultured,passaged, and/or selected in vitro to assess efficacy of an indicatedtreatment. The cells may pluripotent (e.g., induced pluripotent),multipotent, or unipotent stem cells, such as hematopoietic stem cells.Alternatively, the cells can be somatic cells or terminallydifferentiated cells derived from stem cells (e.g., terminallydifferentiated induced pluripotent stem cells).

The monitoring can be used to detect or infer the presence or absence ofa genome edit. In some cases, the monitoring is quantitative, relativelyquantitative, or provides absolute quantitation. The monitoring can bedone using partitioning based assays, such as assays that utilizedigital amplification. The monitoring can be done using droplet digitalassays, such as ddPCR assays. Droplet digital assays for mutationdetection include, e.g., those described in U.S. 2014/0309128. In somecases, the monitoring is done using detection of an inserted HDRtemplate during homology directed repair of a site-specific genomeediting reagent cleavage or nick pair site. In some cases, themonitoring is performed by simultaneous quantification of HDR and NHEJmutations in a sample containing a population of cells that have beencontacted with a genome editing reagent or a genome editing reagent andan HDR template oligonucleotide. In some cases, the monitoring isperformed by massively parallel sequencing, such as targeted or wholegenome massively parallel sequencing.

The testing can be done using tissue, blood, whole blood (or a fractionthereof), plasma, serum, urine, saliva, fecal matter, or any other fluidor substance that can be extracted from a human or animal. The testingcan include monitoring of specific tissue types to determine if a genomeedited cell transfusion has been successful, a percentage of cells fromthe tissue that contain the genome edit, presence or absence of thegenome edit in the sample, or a change or rate of change in the relativeor absolute abundance of the genome edit (or cells containing the genomeedit) in the sample.

In some cases, a threshold (e.g., a percentage based threshold) maycorrelate with therapeutic outcomes. For example, a percentage of genomeedited cells in a sample over or under a threshold may indicate orpredict success of the treatment or guide treatment decisions.

In some cases, non-threshold methods such as linear or logarithmiccorrelation between the number of genome edited cells in a sample andtherapeutic outcome may be obtained or calculated, e.g., to indicate orpredict success of a treatment, monitor treatment progress, or to guidetreatment decisions. Determination of a risk factor for the successfulor unsuccessful treatment with the genome edited cells can be performed.The risk factor can be determined using statistical techniques (i.e.determination of odds ratios). In some cases, the presence or absence ofthe genome edited cells in the sample can be correlated with therapeuticoutcomes or diagnosis or detection of various disease states that areassociated with particular tissue types and with the intent of guidingtreatment decisions.

Also described herein are methods of providing reports or otherinformation to physicians or medical centers of the presence, absence,or quantity of genome edited cells or edited genomes in a sample. Theinformation can be used to guide therapeutic decisions, e.g., theinformation can be used to guide whether there is a need to administermore edited cells, or whether a different edit may be necessary. Theinformation or report can be based on the testing results, meeting ofthresholds, or correlative risk factors.

In some embodiments, a database can be compiled or populated that cancollect and/or store the information (e.g., therapeutic outcomes) ofmany patients' genome editing testing or treatment results. In somecases, the information can be correlated with treatment decisions, suchas administration of additional or alternative genome edited cells orother treatments known in the art (e.g., chemotherapy, such asanti-cancer antibody or antibody drug conjugate therapy).

In some embodiments, a population of genome edited cells can be obtained(e.g., by use of CRISPR/Cas, TALENS, zinc finger nucleases, or othermethods) or provided, the population can be administered to a patient,and then a sample can be obtained from the patient and analyzed to inferthe number of genome edited cells in the patient. In some cases, thenumber of genome edited cells in the patient is compared to a number ofgenome edited cells administered to the patient. In some cases, thenumber of genome edited cells in the patient is compared to a previouslydetermined number of genome edited cells in the patient inferred from apreviously obtained sample. In some cases, the number of genome editedcells in the patient is compared to a subsequently determined number ofgenome edited cells in the patient inferred from a subsequently obtainedsample. An increase or substantial increase in the number of genomeedited cells in the patient, or a lack of a decrease or substantialdecrease in the number of genome edited cells in the patient canindicate treatment success or a likelihood of successful treatment. Adecrease or substantial decrease or a lack of increase or substantialincrease in the number of genome edited cells in the patient canindicate treatment failure or a likelihood of treatment failure.Alternatively, such a decrease or lack of increase can indicateadministration of additional genome edited cells or a change in atreatment (e.g., drug) regimen. In some cases, the rate of increase ordecrease indicates treatment success or failure or indicates a need toadminister additional genome edited cells. In some cases, one of theforegoing methods described herein can further include administeringadditional or alternative genome edited cells to a subject in needthereof, administering cells to a subject in need thereof that comprisea different genome edit than previously administered genome editedcells, or administering a change in a treatment regimen (e.g., a changein drug or dose) to a subject in need thereof.

In some embodiments, a population of genome edited cells can be obtained(e.g., by use of CRISPR/Cas, TALENS, zinc finger nucleases, or othermethods) or provided, the population can be quantified to determine anumber of genome edited cells in the population or the number can bedetermined from a database or other information repository. Thepopulation can then be selected in vitro and the effect of the selectioncan be determined by quantifying a number of genome edited cells in thepopulation after selection. The selection can comprise in vitro culture,in vitro cell culture passaging, or contacting the cells in vitro with adrug or chemotherapeutic.

An increase or substantial increase in the number of genome edited cellsafter selection, or a lack of a decrease or substantial decrease in thenumber of genome edited cells after selection can indicate treatmentsuccess or a likelihood of successful treatment if the cells areadministered to a subject in need thereof. A decrease or substantialdecrease or a lack of increase or substantial increase in the number ofgenome edited cells in the patient can indicate treatment failure or alikelihood of treatment failure if the cells are administered to asubject in need thereof. Alternatively, such a decrease or lack ofincrease can indicate administration of additional genome edited cellsor a change in a treatment regimen (e.g., a change in drug or dose) ifthe cells are administered to a subject in need thereof. In some cases,the rate of increase or decrease indicates treatment success or failure,indicates a need to administer additional genome edited cells, orindicates a change in a treatment regimen (e.g., a change in drug ordose) if the cells are administered to a subject in need thereof. Insome cases, one of the foregoing methods described herein can furtherinclude administering genome edited cells to a subject in need thereof,administering cells to a subject in need thereof that comprise adifferent genome edit than the in vitro selected cells, or administeringa change in a treatment regimen (e.g., a change in drug or dose) to asubject in need thereof.

II. Reaction Mixtures

Described herein is a reaction mixture containing i) a target genomicregion; ii) a DNA dependent DNA polymerase; iii) a forward and a reverseoligonucleotide amplification primer, wherein the forward and reverseprimers hybridize to opposite strands of, and flank, the target genomicregion, and wherein the primers are configured to amplify the targetgenomic region in the presence of the polymerase; iv) a detectablylabeled oligonucleotide reference probe that hybridizes to the targetgenomic region, regardless of the allele (i.e., hybridizes to wild-type,HDR, and NHEJ edited target genomic regions); v) one or more NHEJdrop-off probes; vi) an HDR probe; and optionally vii) an HDR darkprobe. In some cases, one of the one or more NHEJ drop-off probeshybridizes to a sub-region of the target genomic region that overlapswith the hybridization site of the HDR probe. In such cases, the NHEJprobe can substitute for, and render unnecessary, the use of an HDR darkprobe.

In some cases, the reaction mixture is a reaction mixture in a partitionhaving a volume of about 0.001 nL, about 0.005 nL, about 0.01 nL, about0.02 nL, about 0.03 nL, about 0.04 nL, about 0.05 nL, about 0.06 nL,about 0.07 nL, about 0.08 nL, about 0.09 nL, about 0.1 nL, about 0.2 nL,about 0.3 nL, about 0.4 nL, about 0.5 nL, about 0.6 nL, about 0.7 nL,about 0.8 nL, about 0.9 nL, about 1 nL, about 1.5 nL, about 2 nL, about2.5 nL, about 3 nL, about 3.5 nL, about 4 nL, about 4.5 nL, about 5 nL,about 5.5 nL, about 6 nL, about 6.5 nL, about 7 nL, about 7.5 nL, about8 nL, about 8.5 nL, about 9 nL, about 9.5 nL, about 10 nL, about 11 nL,about 12 nL, about 13 nL, about 14 nL, about 15 nL, about 16 nL, about17 nL, about 18 nL, about 19 nL, about 20 nL, about 25 nL, about 30 nL,about 35 nL, about 40 nL, about 45 nL, or about 50 nL.

In some cases, the reaction mixture is in a partition having a diameterof about 0.001 microns, about 0.005 microns, about 0.01 microns, about0.05 microns, about 0.1 microns, about 0.5 microns, about 1 microns,about 5 microns, about 10 microns, about 20 microns, about 30 microns,about 40 microns, about 50 microns, about 60 microns, about 70 microns,about 80 microns, about 90 microns, about 100 microns, about 150microns, about 200 microns, about 300 microns, about 400 microns, about500 microns, about 600 microns, about 700 microns, about 800 microns,about 900 microns, or about 1000 microns. In some cases, the reactionmixture is in a partition having a diameter of less than about 1000microns, less than about 900 microns, less than about 800 microns, lessthan about 700 microns, less than about 600 microns, less than about 500microns, less than about 400 microns, less than about 300 microns, lessthan about 200 microns, less than about 100 microns, less than about 50microns, or less than about 25 microns.

Also described herein are sets of such reaction mixtures. The set cancontain at least 500 reaction mixtures, at least 1000 reaction mixtures,at least 2000 reaction mixtures, at least 3000 reaction mixtures, atleast 4000 reaction mixtures, at least 5000 reaction mixtures, at least6000 reaction mixtures, at least 7000 reaction mixtures, at least 8000reaction mixtures, at least 10,000 reaction mixtures, at least 15,000reaction mixtures, at least 20,000 reaction mixtures, at least 30,000reaction mixtures, at least 40,000 reaction mixtures, at least 50,000reaction mixtures, at least 60,000 reaction mixtures, at least 70,000reaction mixtures, at least 80,000 reaction mixtures, at least 90,000reaction mixtures, at least 100,000 reaction mixtures, at least 200,000reaction mixtures, at least 300,000 reaction mixtures, at least 400,000reaction mixtures, at least 500,000 reaction mixtures, at least 600,000reaction mixtures, at least 700,000 reaction mixtures, at least 800,000reaction mixtures, at least 900,000 reaction mixtures, at least1,000,000 reaction mixtures, at least 2,000,000 reaction mixtures, atleast 3,000,000 reaction mixtures, at least 4,000,000 reaction mixtures,at least 5,000,000 reaction mixtures, at least 10,000,000 reactionmixtures, at least 20,000,000 reaction mixtures, at least 30,000,000reaction mixtures, at least 40,000,000 reaction mixtures, at least50,000,000 reaction mixtures, at least 60,000,000 reaction mixtures, atleast 70,000,000 reaction mixtures, at least 80,000,000 reactionmixtures, at least 90,000,000 reaction mixtures, at least 100,000,000reaction mixtures, at least 150,000,000 reaction mixtures, or at least200,000,000 reaction mixtures.

In some cases, on average about 0.001, 0.005, 0.01, 0.05, 0.1, 0.5, 1,2, 3, 4, or 5 target genomic regions are present in each reactionmixture of the set of reaction mixtures. In some embodiments, at leastone reaction mixture of the set of reaction mixtures contains no targetgenomic regions. In some embodiments, at least about 1%, 2%, 3%, 4%, 5%,6%, 7%, 8%, 9%, or 10% of the reaction mixtures of the set of reactionmixtures contain no target genomic regions.

All patents, patent applications, and other publications, includingGenBank Accession Numbers, cited in this application are incorporated byreference in the entirety for all purposes.

EXAMPLES

The following examples are provided by way of illustration only and notby way of limitation. Those of skill in the art will readily recognize avariety of non-critical parameters that could be changed or modified toyield essentially the same or similar results.

Example 1 Use of Digital PCR for Simultaneous Quantification of HDR andNHEJ Events in Genome-Edited Cells

Materials and Methods

Primers: Forward (F) and reverse (R) primers are designed to generate anamplicon of ˜200-400 bp around the targeted edit site (HDR). The editsite should sit roughly in the middle of this amplicon (˜100-200 bp ofsequence 5′ and 3′ of the intended edit site). Amplification primersdesigned to generate shorter or longer amplicons can also be utilized ifdesired.

Probes:

-   -   Reference Probe: a fluorescently-labeled reference probe (e.g.,        FAM) hybridizes upstream and adjacent to the reverse        amplification primer, or downstream and adjacent to the forward        amplification primer. This reference probe can be used to detect        and count all mixture partitions containing amplicons,        regardless of allele (WT, NHEJ, or HDR). For maximal signal, the        reference probe should be on the same strand as the        amplification primer to which it is adjacent. However, the        reference probe can be designed to hybridize to either strand of        the amplicon.    -   HDR Probe: a second fluorescently-labeled probe is designed to        hybridize to a genomic region overlapping the HDR edit site. The        HDR and reference probes can be labeled with the same        fluorescent label, or with different fluorescent labels.    -   HDR Dark Probe: a second “dark” oligo probe, which includes a        non-extendible 3′ end and no fluorophore label, is designed that        is nearly identical in sequence to the HDR probe, except for the        HDR edited position. In the Dark Probe, this position        complements the wild-type (non-edited) sequence. The purpose of        this probe is to minimize cross-reactivity of the HDR Probe with        wild-type DNA. This improves separation between WT and HDR        cluster groups in the 2D plot. In some assay designs, a HDR dark        probe may not be necessary at all. In some cases, an NHEJ        drop-off probe that directly competes with the HDR probe for        binding to a wild-type sequence can compensate for the lack of        an HDR dark probe.    -   NHEJ Drop-off Probe: The NHEJ drop-off probe indicates a        mutation (NHEJ) event when probe signal is lost, not gained. It        is a negative signal. The probe is designed to hybridize to a        wild-type sequence at the site where DNA is targeted for cutting        by the specific cutting system (e.g., CRISPR-Cas9 cuts 3-5 bases        5′ upstream of the PAM NGG site. When that wild-type sequence is        mutated, for example by an indel caused by NHEJ, the probe        “drops off” and cannot bind; therefore, loss of fluorescence        amplitude (e.g., HEX if the NHEJ drop-off probe is labeled with        HEX) is indicative of an NHEJ event (with only the FAM reference        signal remaining to mark this allele). In the case of genome        editing strategies with more than one cut site (e.g.,        Cas9-Nickase), more than one NHEJ drop-off probe can be required        to assay all potential NHEJ mutation sites. Alternatively, a        longer NHEJ drop-off probe can be utilized to hybridize to (and        thus interrogate) multiple potential NHEJ sites.        Exemplary probe and primer compositions for quantifying NHEJ        (SEQ ID NOS:9, 15 and 16) and HDR (SEQ ID NO:10) mutations at        the genomic site RBM20 R636S (SEQ IDS NOS:12, 32, 33-13, 34 and        35, respectively, in order of appearance) with three different        genome editing reagents (CRISPR-Cas9, Cas9-Nickase, and        dCas9-FokI) are illustrated in FIG. 6.

Assay composition: a reaction mixture is formed at a final 1×concentration containing the following:

-   -   Forward and Reverse amplification primers: each at 900 nM (SEQ        ID NOS:8 and 14)    -   Reference Probe: 250 nM (SEQ ID NO:11)    -   HDR Probe: 250 nM    -   HDR dark probe: 500 nM    -   NHEJ Drop-off Probe (s): 250 nM each

Digital PCR Reaction Setup:

-   -   The digital PCR assay is set up using standard protocols known        in the art. For example, 100 ng of genomic DNA sample (˜30,000        human genome equivalents) are combined with supermix (ddPCR        Supermix for Probes (no dUTP)), assay buffer, 2 U of a        restriction endonuclease that does not cut the wild-type or HDR        edited amplicon (e.g., a six-cutter (or longer) like HindIII-HF)        and water. The components are thoroughly mixed (e.g., by        vortexing). Partitioning is performed (e.g., droplets are        generated) and the partitions are thermal cycled.    -   Thermal cycling protocol: to accommodate long amplicons, a        3-step thermal cycling protocol can be used. In some cases, a 2        step protocol is adequate. A 3-step protocol with a 2 minute        extension should adequately cover amplicons up to 1 kB.        Amplicons are generated according to the following exemplary        protocol:        -   95° C., 10 min        -   94° C. 30 sec        -   Anneal Temp (as defined by Temp Gradient), e.g., 59° C., 1            min        -   72° C., 2 min        -   Go to step 2, 39×        -   98° C., 10 min        -   12° C., hold    -   Detect hybridization of fluorescently-labeled probes in the        partitions    -   Using this method with droplet digital PCR (ddPCR), the maximal        number of NHEJ mutant copies will be read when 0.6 to 1.6 copies        per droplet (cpd) of genomic DNA is loaded per well. This        represents ˜40 ng-100 ng of human genomic DNA per well. 1 cpd        loading (66 ng) is optimal mathematically, but real-world        factors like sample integrity and assay performance will also        influence performance at 1 cpd loading. For example, at higher        loading, separation between signals from droplets containing        wild-type target genomic region and HDR-edited target genomic        region can be reduced. Generally, 100 ng of genomic DNA/well is        an amount that will provide robust and accurate quantification        by ddPCR, but running a sample dilution series using appropriate        controls like gblocks can identify the optimal loading for a        given assay.

Analysis: Cluster groups are first identified. For example, clustergroups can be identified in Quantasoft by lasso thresholding. When twochannel detection is utilized, and the HDR and reference probes arelabeled with the same fluorescent label (e.g., FAM), the fluorescentsignal in partitions containing HDR-edited target genomic regions is anadditive function of the signal from both the HDR and the referenceprobes.

-   -   To correctly lasso the given clusters, the positive control        wells can be used to identify correct cluster positions and        amplitudes. An example positive control well contains wild-type        (WT) DNA at same loading regimen as the samples, plus gblock        synthetic template controls at a given percent (e.g., 5% mutant        copies). Another example positive control is a well that        contains only WT DNA (FIG. 7). One can thus use the control well        cluster positions to assign clusters in the sample wells.    -   Using the described assay strategy, the cluster classes will        appear and can be lasso thresholded in this manner:        -   Ch1+Ch2+ droplets: WT+WT/NHEJ droplets        -   Ch1+Ch2−: NHEJ        -   Ch1++Ch2+: HDR+HDR/WT droplets        -   Ch1-Ch2−: negatives    -   Note in this example, a given cluster might contain more than        one droplet group (as indicated by oval overlays, approximately        (see FIG. 8)). The analysis method takes this fact into account        when computing a concentration call for a given species, such as        NHEJ alleles. This enables accurate quantification even at        higher loading regimens.    -   The concentration calls (copies/μL) of HDR, NHEJ, and wild-type        alleles can be determined per well, including with 95%        confidence intervals. From these numbers, percent NHEJ and        percent HDR as well as ratio HDR:NHEJ per sample per well can be        easily derived.

Quantification:

-   The standard formula for ddPCR quantification is:

$c = {{- {\ln\left( \frac{N_{neg}}{N_{total}} \right)}}/V_{{drople}t}}$

-   where-   N_(neg)=the number of droplets that do not contain the species of    interest-   N_(total)=the total number of droplets

For the assay described here, some of the droplet populations cannoteasily be separated (e.g., the wild-type (WT) and NHEJ+WT populations).To quantify in this case, an appropriate subset of droplets is used tocalculate N_(neg) and N_(total).

Definitions:

-   -   N_(empty)=the number of droplets in the cluster labeled “empty”        (black)    -   N_(NHEJ)=the number of droplets in the cluster labeled “NHEJ”        (blue)    -   N_(WT+)=the total number of droplets in the clusters labeled        “WT” and “NHEJ+WT” (green)    -   N_(HDR+)=the total number of droplets in the clusters labeled        “HDR” and “HDR+WT” (orange) plus the number of droplets in the        clusters “HDR+NHEJ” and “HDR+NHEJ+WT” (too few droplets to be        seen clearly in data)    -   N=the total number of observed droplets

For NHEJ quantification:

N _(total) =N _(empty) +N _(NHEJ)

N_(neg)=N_(empty)

For HDR quantification:

N _(total) =N _(empty) +N _(NHEJ) +N _(WT+) +N _(HDR+)

N _(neg) =N _(empty) +N _(NHEJ) +N _(WT+)

For WT quantification:

N _(total) =N _(empty) +N _(NHEJ) +N _(WT+)

N _(neg) =N _(empty) +N _(NHEJ)

Example 2 Simultaneous Quantification of HDR and NHEJ Events Induced bySequence—Specific Nucleases Using ddPCR Introduction

Sequence-specific nucleases such as TALENs and the CRISPR/Cas9 systemactivate the DNA repair pathways of non-homologous end joining (NHEJ) orhomology-directed repair (HDR) at target sites. Although error-prone,NHEJ is useful for disrupting gene function. For many applications HDRis more desirable than NHEJ, since HDR utilizes homologous donor DNA toproduce precise gene repair. Since NHEJ and HDR involve different repairenzymes, it is conceivable that conditions could be achieved with highHDR and low NHEJ. However, methods for altering the balance between NHEJand HDR are elusive, since we lack a rapid sensitive assay to quantifyNHEJ and HDR at endogenous genomic loci. To overcome this hurdle, amethod is described herein to detect NHEJ and HDR simultaneously basedon droplet digital PCR (ddPCR) and fluorescent oligonucleotide probesspecific to wild-type (WT), NHEJ, and HDR alleles. The method can allowtesting of multiple conditions for genome editing including differenttypes of sequence-specific nucleases and donor DNAs in order to tilt thebalance between NHEJ and HDR towards the desired repair pathway.

Materials and Methods

The QX100 Droplet Digital PCR system (Bio-Rad) was utilized for ddPCR.TALENs were constructed using the Voytas laboratory's Golden Gateassembly system (Cermak et al., Nucleic Acids Res. 39, e82, 2011), andthe MR015 backbone vector (gift from M. Porteus and M. Randar, StanfordUniversity). Guide RNAs (gRNAs) were constructed using the Zhanglaboratory's pX335 (nickase) and pX330 (nuclease) (Ran et al., Cell 154,1380-1389, 2013). Probes and primers were from Integrated DNATechnologies.

HDR and NHEJ in Single Cells

FIG. 9A-B illustrate a strategy for targeting the GRN locus for pointmutagenesis in human induced pluripotent stem (iPS) cells. A pair ofgRNAs for Cas9 nickase were designed to introduce a C>T point mutationin the GRN locus (FIG. 9A). The wild-type C (SEQ ID NO:17-18) and mutantT (SEQ ID NO:20-21) residues are highlighted by rectangles. The nicksites are indicated by triangles. Sequences of an isolated iPS cellclone were aligned to the WT sequence (SEQ ID NOS:22-24) (FIG. 9B). Thisclone had the C>T point mutation (SEQ ID NO:23) and a 24-bp deletion(SEQ ID NO:24). These results indicate that HDR and NHEJ can happen insingle cells, ending up in compound heterozygous mutant cells.

Design of Targeting and Detection of HDR

FIG. 10A-B illustrate a strategy for targeting and detection of homologydirected repair (HDR) mutations in cells. FIG. 10A depicts the design ofthe TALEN, CRISPR/Cas9, and TaqMan PCR systems for mutagenesis at theRBM20 locus. A pair of TALENs and a pair of gRNAs (SEQ ID NOS:25-26)were designed. A 60-nt donor oligonucleotide (SEQ ID NO:27) that has aC>A mutation as compared to the wild-type sequence (SEQ ID NOS:28-29)was designed to act as a template for HDR repair, thereby introducingthe C>A mutation into a cell (SEQ ID NOS:30-31). An allelic-specificTaqMan PCR assay, designed to detect specific edits induced by HDR butnot detect NHEJ, is also depicted. In this assay, a carboxyfluoresceinlabeled probe complementary to the wild-type sequence is utilized todetect the wild-type sequence and a VIC labeled probe complementary tothe HDR mutant sequence encoded by the donor oligonucleotide is utilizedto detect wild-type and HDR mutations respectively. As shown in FIG.10B, the following different nuclease platforms that were compared inthis study: CRISPR/Cas9, CRISPR/Cas9 nickase, and TALEN to compare theiractivities to induce HDR and NHEJ.

ddPCR Assay to Detect Only HDR

FIG. 11A-C illustrate the basics of a ddPCR assay for detection of HDRonly. The reaction is partitioned into over 10,000 nanoliterwater-in-oil droplets. Each droplet contains all the PCR reagents and afew or no copies of template DNA. Thermal cycling amplifies the FAM,VIC, or both signals in each droplet depending on the templates present.Finally, the signals are measured in individual droplets, allowingdetection of rare templates. Concentration is reported in copies/μl.FIG. 11B depicts a two-dimensional plot of ddPCR for a 1:1 mixture ofthe WT and HDR allele. The numbers of negative (black), FAM+ (WTallele), VIC+ (HDR allele), and FAM and VIC double+ (WT and HDR alleles)droplets are used to calculate the concentrations of the alleles. FIG.11C depicts ddPCR data for a dilution series of the HDR allele and theWT allele. Plasmids with the WT− or HDR allele were mixed at differentratios. ddPCR can be used to robustly detect 0.1% or fewer HDR allelesin a population of WT alleles.

ddPCR Assay to Detect HDR and NHEJ

FIG. 12 illustrates a design for a ddPCR assay for simultaneousdetection of HDR and NHEJ. An assay for detection of Cas9 mutagenesis isshown as an example. In this example, the assay utilizes aFAM-conjugated reference probe (Ref. probe) that detects a conservedsequence in the amplicon that is distal from the mutagenesis site, aFAM-conjugated HDR probe with the point mutant allele sequence thatdetects HDR events, a HEX-conjugated NHEJ probe located at the cut sitewith the conserved sequence that detects NHEJ events (or HDR events inthe NHEJ probe binding region) when it fails to bind, and a Darknon-extendable competitor probe with the conserved sequence at the HDRsite but lacking fluorophore. After mutagenesis to induce the RBM20point mutation shown in FIG. 10A-B, if no modification was left, the WTallele would stay intact, giving FAM+ and HEX+ because the Ref probe andNHEJ probe bind to the WT allele. The Dark probe competes with HDR probepreventing HDR probe from binding to the WT allele. If HDR occurred tointroduce the C>A conversion, HDR probe would bind to the created mutantallele instead of Dark probe, so that the HDR allele would be higheramplitude FAM++ and HEX+ as the HDR allele has two binding sites for FAMprobes. If an NHEJ mutation occurs, the NHEJ probe would lose itsbinding site, but the Ref probe would remain bound, so that the NHEJallele would be detected as be FAM+ and HEX−. As a result, threedifferent types of alleles can be detected as different populations inddPCR. Assays for Cas9 nickase and TALENs are shown in the boxed portionof FIG. 12. Since a pair of Cas9 nickases are used, the Cas9 nickaseassay uses two NHEJ probes.

Validation of Assay for Simultaneous Detection of HDR and NHEJ Mutations

FIG. 13A-D depict validation data for a ddPCR assay to detect HDR andNHEJ induced mutations from a Cas9-based genome editing reagent withsynthesized DNA (gBlock). Genomic DNA from WT HEK293 cells without (FIG.13A) and with (FIG. 13B) 5% gBlock DNA of HDR and NHEJ alleles wereanalyzed by the Cas9 probe mixture shown in FIG. 12 (Ref., HDR, NHEJ,and Dark probes). All alleles were FAM+ and HEX+ in the WT genomic DNAsample, but HDR (brown) and NHEJ (blue) alleles were detected when thegBlock DNA was added. Measurement of the quantitative performance of theHDR and NHEJ assay with synthesized DNA shows that the limit ofquantification was around 0.1% for detection of NHEJ mutations and lowerthan 0.04% for detection of HDR mutations (FIG. 13C). HEK293 cells weretreated with Cas9 to induce HDR as shown in FIG. 10A, and their genomicDNA was analyzed. Both HDR+ and NHEJ+ populations were observed (FIG.13D).

Comparison of Different Nucleases

HEK293 cells or human iPS cells were transfected with RBM20 TALENs,Cas9, or Cas9 nickases together with an RBM20 R636S single strand oligoDNA donor to introduce the C>A mutation as shown in FIG. 10A-B. GenomicDNA isolated from these cells were analyzed by using the probe andprimer sets described in FIGS. 12 and 13 to detect HDR and NHEJmutations. The copy numbers of the HDR and NHEJ alleles normalized tothat of the WT allele are shown in FIG. 14, left graph. FIG. 14, rightgraph shows the data for HDR mutations only. All the nucleases inducedmuch more NHEJ than HDR in both HEK293 cells and iPS cells.

Conclusion

Described herein is a highly sensitive, quantitative, and rapid methodto simultaneously detect HDR and NHEJ events. By using this method,sequence-specific nucleases are shown to induce much more NHEJ than HDR.This method can enable a facile search for improved genome editingconditions. For example, the assay can be used to identify conditionsthat increase the generation of HDR mutations while minimizing thegeneration of NHEJ mutations.

What is claimed is:
 1. A plurality of mixture partitions having anaverage volume, wherein the mixture partitions comprise: i) a targetgenomic region from cells that have been contacted with: (1) asite-specific genome editing reagent configured to cleave or nick DNA inthe target genomic region and (2) an HDR template nucleic acid; ii) aDNA-dependent DNA polymerase; iii) a forward and a reverseoligonucleotide amplification primer, wherein the forward and reverseprimers hybridize to opposite strands of, and flank, the target genomicregion, and wherein the primers are configured to amplify the targetgenomic region in the presence of the polymerase; iv) a detectablylabeled oligonucleotide reference probe, wherein the reference probehybridizes to a wild-type target genomic region, a target genomic regioncontaining an HDR mutation introduced by homology directed repair of DNAdamage from the site specific genome editing reagent, and a targetgenomic region containing an NHEJ mutation introduced by NHEJ repair ofDNA damage from the site specific genome editing reagent; v) adetectably labeled oligonucleotide HDR probe, wherein the HDR probehybridizes to the target genomic region containing the HDR mutation; andvi) a detectably labeled oligonucleotide NHEJ drop-off probe, whereinthe NHEJ drop-off probe hybridizes to the wild-type target genomicregion, and wherein the NHEJ drop-off probe does not hybridize to thetarget genomic region containing the NHEJ mutation.
 2. The plurality ofpartitions of claim 1, wherein the reference and HDR probes comprise thesame detectable label.
 3. The plurality of partitions of claim 1,wherein the HDR probe does not hybridize to the wild-type target genomicregion.
 4. The plurality of partitions of claim 1, wherein the NHEJ dropoff probe does not hybridize to the target genomic region containing theHDR mutation.
 5. The plurality of partitions of claim 1, wherein theNHEJ drop off probe hybridizes to the target genomic region containingthe HDR mutation.
 6. The plurality of partitions of claim 1, wherein theDNA dependent DNA polymerase comprises 5′ to 3′ exonuclease activity 7.The plurality of partitions of claim 1, wherein the plurality of mixturepartitions further comprise an HDR dark oligonucleotide probe, whereinthe HDR dark probe comprises a 3′ end that is not extendable by the DNApolymerase, and wherein the HDR dark probe competes for hybridization ofthe HDR probe to the wild-type target genomic region.
 8. The pluralityof partitions of claim 1, wherein the site-specific genome editingreagent is a CRISPR-Cas9 reagent, a TALEN, or a Zinc-Finger Nuclease. 9.The plurality of partitions of claim 1, wherein the plurality of mixturepartitions comprise a plurality of structurally different detectablylabeled oligonucleotide NHEJ drop-off probes, wherein the plurality ofstructurally different NHEJ drop-off probes hybridize to differentsub-regions of the wild-type target genomic region.
 10. The plurality ofpartitions of claim 9, wherein the plurality of structurally differentNHEJ drop off probes do not hybridize to NHEJ mutated sub-regions. 11.The plurality of partitions of claim 9, wherein the plurality ofstructurally different NHEJ drop off probes do not hybridize to NHEJmutated sub-regions and do hybridize to target genomic regionscontaining an HDR mutation.
 12. The plurality of partitions of claim 1,wherein the partitions are aqueous droplets that are surrounded by animmiscible carrier fluid.
 13. The plurality of partitions of claim 1,wherein the plurality comprises at least 500 partitions.